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Sustaining immunity during starvation in bivalve mollusc: A costly affair
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Sustaining immunity during starvation in bivalve mollusc: A costly affair Elizabeth Mahapatra a , Dishari Dasgupta a , Navodipa Bhattacharya a , Suvrotoa Mitra a , Debakana Banerjee a , Soumita Goswami a , Nabanita Ghosh a , Avijit Dey a , Sudipta Chakraborty a,b,∗ a b
Parasitology and Immunology Laboratory, Department of Zoology, Maulana Azad College, 8, Rafi Ahmed Kidwai Road, Kolkata 700013, West Bengal, India Department of Zoology, Government General Degree College, Keshiary, Paschim Medinipur 721133, West Bengal, India
a r t i c l e
i n f o
Article history: Received 14 September 2016 Received in revised form 17 February 2017 Accepted 17 February 2017 Available online xxx Keywords: Starvation Bivalve Phagocytosis Nitric oxide Haemocytes
a b s t r a c t Complete or partial depletion of resource in a freshwater habitat is a common phenomenon. As a consequence, aquatic fauna including bivalve molluscs may be exposed to dietary stress on a seasonal basis. Haemocyte based innate immune profile of the freshwater mollusc Lamellidens marginalis (Bivalvia: Eulamellibranchiata) was evaluated under starvation induced stress for a maximum period of 32 days in a controlled laboratory condition. During starvation, the bivalve haemocytes maintained a homeostasis in phagocytic efficacy and nitric oxide generation ability with respect to the control. The mollusc maintained a significantly high protein content in its haemolymph and tissues under the nutritional stress with respect to the control. The dietary stress had no significant impact on the activity of digestive tissue derived ␣-amylase till sixteenth day but by 32 days the enzyme activity went down significantly. The histopathological profile revealed that the bivalve was adapted to maintain a steady immune profile by incurring degeneration of its own tissue structure. The total haemocyte count surged significantly till 16 days but differed insignificantly with respect to the control at 32 days implying probable haematopoietic exhaustion. The study reflects the instinctive urge of the bivalve to maintain immune physiology at heavy metabolic cost under nutrient limited condition. © 2017 Elsevier Ltd. All rights reserved.
1. Introduction Molluscs, including the bivalves, occupy an important position in the freshwater ecosystem of the tropics by stabilising it against natural as well as anthropogenic stresses (Dudgeon et al., 2006; Chakraborty et al., 2012; Fritts et al., 2015). The filter-feeding habits of molluscs, including bivalves, regulate the productivity and health of an aquatic environment (Officer et al., 1982; Newell, 2004). The sensitivity of the bivalves to the environmental stressors is manifested through the altered structural and functional attributes of their circulating haemocytes (Chakraborty et al., 2008; Calisi et al., 2008; Chakraborty and Ray, 2009; Martins and Costa, 2015; Matozzo, 2016) and tissues (Belcheva et al., 2011; Chakraborty et al., 2013; deOliveira et al., 2016). The circulating haemocytes of the molluscs constitute a vital component of its defence strategy
∗ Corresponding author at: Parasitology and Immunology Laboratory, Department of Zoology, Maulana Azad College, 8, Rafi Ahmed Kidwai Road, Kolkata 700013, West Bengal, India. E-mail address: [email protected] (S. Chakraborty).
and nutrient dynamics (Feng et al., 1977; Cheng, 1981). Phagocytosis of non-self particulates and generation of reactive nitrogen intermediates (RNIs) like nitric oxide are evolutionarily conserved strategies which are employed by the molluscan haemocytes to combat pathogenic insults (Araya et al., 2009; Chakraborty et al., 2009; Park et al., 2012). Studying the fluctuations of the molluscan haemocyte morphotypes with relation to diverse stressors seems important in order to perceive the environmental changes (Pamparinin et al., 2002). Bivalves thus have become a dependable model for environment biomonitoring (Sanders, 1993; FernándezTajes et al., 2011; Fritts et al., 2015) and their immunological profile represents the health of their resident environment (Sauvé et al., 2002; Girón-Pérez, 2010; Bhunia et al., 2016). Introduction of bivalve to aquasystems deprived of unionid fauna is a popular environment management strategy to incorporate trophic stability in oligotrophic environment (Patterson et al., 1999; Vaughn and Hakenkamp, 2001; Xu et al., 2008). Their inclusion in an existing population may improve its genetic diversity (Cope and Waller, 1995; Szumowski et al., 2012). The relocated bivalve population may thus be temporarily subjected to a con-
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dition of complete food deprivation due to the founder effect (Hoftyzer et al., 2008; Gilg et al., 2013) or the reinforced population may suffer from partial food deprivation due to augmented competition (Wacker and von Elert, 2002; Guernic et al., 2015). Partial or complete starvation may alter the biochemical composition of bivalves (Whyte et al., 1990), decrease their metabolic activity (Rodhouse and Gaffney, 1984) and change the haemocyte defence capacity (Butt et al., 2007). Lamellidens marginalis (Mollusca; Bivalvia) is a common bivalve mollusc of the freshwater aquasystems of eastern India including West Bengal (Fig. 1a). It is often used as a dietary supplement for poultry as well as human feed in rural West Bengal and adjoining states (Chakraborty et al., 2008; Madhyastha et al., 2010). It is a potential culturable aquacrop since pearls are naturally formed in the bivalve (Barik et al., 2004; Madhyastha et al., 2010). Its physiology has exhibited dose-responsiveness to diverse environmental pollutants like cadmium, chromium, arsenic (Das and Jana, 2004; Satyaparameshwar et al., 2006; Chakraborty et al., 2008, 2012, 2013) and stressors like oxygen and ammonia (Indira and Chetty, 1994; Das and Jana, 2003) which validates its prospective role in environment biomonitoring. However, the effect of starvation on the immunological profile of the haemocytes with relation to the morphological stability of tissue has not been investigated in details in molluscan model. The study was designed to understand the alterations in total count and selected immunological profiles of the haemocytes of L. marginalis subjected to dietary stress for a defined time span under controlled laboratory conditions. Histopathological study of selected organs of the starved molluscs would be helpful to characterise the effect of the stressor on its tissue structure. The data would be useful to analyse the impact of starvation induced stress on aquatic biota which would assist in formulation of a sustainable strategy of conservation of freshwater aquasystems.
2. Materials and methods
2.3. Enumeration of total haemocyte count (THC) Haemolymph was collected aseptically from the heart and posterior adductor muscle of the bivalves (Brousseau et al., 1999; Ahmad et al., 2011) in chilled glass vials. The THC was recorded separately from the haemolymph collected from the heart and posterior adductor muscle using Neubauer’s haemocytometer (Chakraborty et al., 2008). The enumeration of THC was carried out on haemolymph sampled from each of the 10 bivalves per experimental batch and the experiment was repeated for 5 times.
2.4. Enumeration of phagocytic index Cultured baker’s yeast (Saccharomyces cerevisiae) was killed by boiling for 1 h and the resulting cell suspension was washed three times in pH 7.4 TBS/Ca2+ (20 mM Trizma base, 77 mM NaCl, 10 mM CaCl2 ) by centrifugation at 650 × g for 10 min. The washed cells were then suspended at a concentration of 107 cells/ml in Grace’s Insect Medium (Himedia). The phagocytic efficiency of the haemocytes was examined by challenging them with yeast suspension in a ratio of about 1:10 in vitro over slide. To the adherent monolayer of haemocytes, 1 l of yeast (1 × 107 cells/ml) was added and incubated at 26 ◦ C in a humid chamber for 2 h. After incubation, the monolayer was washed with sterile snail saline (5 mM HEPES, 3.7 mM NaOH, 36 mM NaCl, 2 mM KCl, 2 mM MgCl2 ·2H2 O, 4 mM CaCl2 ·2H2 O; pH 7.8) (Adema et al., 1991), stained with Giemsa’s stain and observed under microscope (Axiostar Plus, Zeiss). A negative control for the assay was set using a known phagocytic inhibitor sodium azide (2%). More than 200 fields were examined for each slide and data was recorded to enumerate phagocytic index (PI), where PI = [(engulfed particles/phagocytic cells) × (phagocytic cells/total cells) × 100] (Elssner et al., 2004). The enumeration of PI was carried out on haemolymph sampled from each of the 10 bivalves per experimental batch and the experiment was repeated for 5 times.
2.1. Collection and maintenance of animals Fresh specimens of L. marginalis measuring 5 cm (±0.342) × 3.5 cm (±0.164) were sampled from the selected freshwater ponds of the district of 24-Parganas (South) of West Bengal in India. The collected animals (90 bivalves for each experiment) were transported to the laboratory and acclimated for 7 days in well-aerated tanks (90 cm × 90 cm × 60 cm) in batches of 15 per tank. They were provided with a feed of chopped lettuce leaves. The water of the tanks was replaced every 12 h to remove unutilized food and metabolic wastes. The average dissolved oxygen content and hardness of the water in the storage tanks were maintained at 14.1 mg/l and 457 mg/l respectively after Raut (1991). The water, soil sediments from the site of animal collection and bivalves from the sampling sites were tested periodically to trace heavy metal and metalloid contamination which yielded negative results.
2.5. Estimation of nitrite generation in haemocytes The estimation of the generation of NO was carried out on haemolymph pooled separately from the heart and posterior adductor muscle of the 10 bivalves per experimental batch. The generation of NO in the haemocyte was measured with Griess reagent modified after Green et al. (1982). The density of haemocytes was adjusted to 106 cells/ml and 1 ml of the haemocyte suspension was incubated with equal volume of Griess reagent (1% sulphanilamide, 0.1% naphthyl ethylenediamine dihydrochloride and 5% orthophosphoric acid) at 26 ◦ C for 30 min in a humid chamber. The absorbance was recorded in a spectrophotometer (Shimadzu 1800) at 550 nm against a standard blank. The generation of nitrite was determined using a standard curve of sodium nitrite. The generation of NO was expressed in terms of formation of nitrite as M/min/106 cells and the experiment was repeated for 5 times.
2.2. Exposure to stress 2.6. Preparation of tissue lysates Six separate batches comprising 10 bivalves per batch were subjected to starvation for a time span of 1, 2, 4, 8, 16 and 32 days (D) respectively. Each batch of the bivalves was maintained in wellaerated glass water tanks of 20 l capacity. One similar batch of bivalves was maintained with standard feed as control. The water of the tanks was replaced every 12 h to remove unutilized food and metabolic wastes. The experimental bivalves were monitored for possible visible health abnormalities and death. The experiments were repeated for 5 times (n = 5).
The tissue samples were dissected out of the heart, gill and digestive organ of the 10 bivalves per experimental batch and pooled separately before washing in chilled SSS. The tissue samples were weighed, homogenized in SSS and washed twice by centrifugation at 3000 rpm for 20 min at 4 ◦ C. The final pellet was resuspended with 1 ml of 0.1%. Triton X-100 and incubated under ice for 30 min. The suspensions were then centrifuged at 8000 rpm for 30 min at 4 ◦ C and the supernatants were collected and stored
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Fig. 1. (a) Control L. marginalis, (b) L. marginalis starved for 32 D exhibiting melanized shell and (c) dynamics of total haemocyte count in the haemolymph from heart and foot muscle of L. marginalis under starvation for a maximum time-span of 32 D (data presented as mean ± S.D.; n = 5; *P < 0.05).
in chilled vials at 4 ◦ C for not more than 1 h before being used for experimentation.
maltose was used to quantitate the optical densities and the kinetics of activity of ␣-amylase was expressed as rate of generation of maltose (M/min). The experiment was repeated for 5 times.
2.7. Estimation of total protein in haemolymph and tissue The estimation of the total protein of the haemolymph and tissue lysates was carried out spectrophotometrically (Shimadzu 1800) after Lowry et al. (1951) using a standard curve of bovine serum albumin. The experiment was repeated for 5 times. 2.8. Estimation of ˛-amylase activity in digestive tissue The kinetics of ␣-amylase (EC 3.2.1.1) activity was assayed by estimating the rate of generation of reducing sugars from starch by 3, 5-dinitrosalicyclic acid (DNS) method modified after Bertrand et al. (2004). In the process, 1 ml of the digestive tissue lysate was incubated with 1 ml of 1% starch solution (prepared in SSS) in 3 batches of test tubes. The reaction mixture in each of the three batches of test tubes was incubated in a humid chamber at 26 ◦ C for 10 min, 20 min and 30 min respectively. A blank consisting of 1 ml of 1% starch solution and 1 ml of SSS was incubated in identical conditions. The reaction was terminated by adding 2 ml of DNS reagent in each test tube and then incubating the tubes in a boiling water bath for 10 min. The test tubes were allowed to cool and the absorbance of the final reaction mixtures was spectrophotometrically (Shimadzu 1800) measured at 540 nm. A standard curve of
2.9. Histopathology of vital tissues The tissue samples were collected from the mantle, gill, labial palp, heart, foot muscle, gut and gonad of the control and 32 D starved animals. After autopsy, the tissue samples were washed in SSS and further processed for standard histological preparations after Chakraborty et al. (2010). The tissue sections were stained with haematoxylin-eosin, mounted in DPX, studied under microscope (Axiostar Plus, Zeiss). The documentation of tissue dimensions was carried out with an oculometer calibrated against a stage micrometre (Erma, Japan) after Carballal et al. (1997). 2.10. Statistical analysis The mean values of the experimental control and starved animals were compared using factorial Analysis of Variance (ANOVA). Pearson’s correlation coefficient was calculated to analyse relationship amongst the observed parameters of the bivalve along the entire life-span of starvation induced stress. SPSS Statistics versions 16 software was used for processing of the data (p < 0.05) and the data were expressed as mean ± standard deviation (S.D.).
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3. Results
3.6. Histopathology
3.1. THC
All histopathological exhibits were sampled from the control and 32 D starved bivalves. The mantle of the starved bivalve revealed intense haemocyte infiltration, development of granulomatous tissue and formation of vacuole in the adipocytes (Fig. 6a and b). The starved gill and labial palp of the bivalve also exhibited accumulation of hyperchromatic haemocytes within the inner lamellar space and subepithelial connective tissue. The central water channel of the starved gill was dilated at places and the gill filaments and the ciliated epithelium of the labial palp appeared to have suffered shrinkage (Fig. 6c–f). The starved bivalve exhibited intense inflammatory tissue damage in ventricular and the pericardial muscle of the heart (Fig. 6e and f), loss of compactness in the somatic muscle of the foot due to development of numerous vacuoles (Fig. 6g and h) and thinning of the intestinal wall with dilation of its lumen (Fig. 6i and j). The female gonadal tissue of the bivalve seemed least affected by starvation and exhibited normal tissue architecture for the acini and germinal duct (Fig. 6k). The oocytes appeared to be healthy with distinct vitelline layer and perivitelline space (Fig. 6l). The male gonadal tissue of the starved bivalve apparently maintained tissue composure although the density of the spermatids was visibly low with respect to the control animals (Fig. 6o and p).
The THC in the haemolymph collected from the heart of the bivalves exhibited a significant increase by 4 D of starvation as 10.34 × 106 ± 1.005 cells/ml with respect to the control value of 0.4 × 106 ± 0.134 cells/ml. Eventually the THC attained a value as 0.33 × 106 ± 0.324 cells/ml by 32 D without any significant difference with respect to the control (Fig. 1c). The THC in the haemolymph collected from the posterior adductor muscle of the bivalves exhibited identical pattern with the progression of starvation induced stress till 32 D with the highest count recorded on 4 D as 1.4 × 106 ± 0.08 cells/ml with respect to the control value of 0.271 × 106 ± 0.012 cells/ml (Fig. 1c; Table 2). 3.2. Phagocytic index The haemocytes of the bivalve exhibited varying phagocytic ability under exposure of starvation induced stress (Fig. 2a–c). The PI of the haemocytes collected from haemolymph of the heart of the bivalves exhibited no significant alteration with respect to the control value of 145 ± 12.7 with the only exception being on 2 D of starvation when the PI value was enumerated as high as 316 ± 19.432 (Fig. 2d). On the contrary, on 2 D of starvation, the PI value of the haemocytes collected from the posterior adductor muscle of the bivalve exhibited a significant drop to 113 ± 15.318 with respect to the control value of 467 ± 13.675. However, the PI value exhibited no significant difference by 32 D of starvation with respect to the control (Fig. 2d; Table 2). 3.3. Nitrite generation in haemocytes The generation of nitrite by the haemocytes collected from haemolymph of the heart of the bivalves remained significantly lower from 2 D till 8 D of starvation with respect to the control. However, the generation of nitrite attained insignificant deviation with respect to the control by 32 D of starvation (Fig. 3). The generation of nitrite by the haemocytes collected from haemolymph of the posterior adductor muscle of the bivalves exhibited identical pattern of fluctuation with respect to the control values. However, the generation of nitrite attained insignificant deviation with respect to the control by 16 D of starvation (Fig. 3; Table 2). 3.4. Total protein in haemolymph and tissue The content of total protein in the haemolymph from heart and posterior adductor muscle of the bivalve exhibited significant increase by 16 D and 32 D of starvation with respect to the control (Fig. 4a). The protein content of the heart muscle, gill and digestive gland tissue also increased in identical pattern by 32 D of starvation as 31.25 ± 0.832 mg/gm, 10 ± 2.041 mg/gm and 33.3 ± 3.321 mg/gm with respect to the control values of 8.670 ± 0.613 mg/gm, 2.560 ± 1.102 mg/gm and 1.037 ± 0.112 mg/gm respectively (Fig. 4b; Table 2). 3.5. Amylase activity in digestive tissue The kinetics of generation of maltose from starch due to amylase activity by the digestive tissue lysate from the bivalve exhibited a significant reduction by 32 D of starvation as 0.033 ± 0.002 M/min and 0.01 ± 0.006 M/min at 10 and 30 min with respect to the control values of 0.122 ± 0.002 M/min and 0.031 ± 0.004 M/min at 10 and 30 min respectively (Fig. 5; Table 2).
4. Discussion The study depicts the primal instinct of the bivalve mollusc to attain and maintain physiological homeostasis, only at a cost. The surge in the THC with the progression of starvation till eighth day probably reflects the innate response of the bivalve to adjust with the stress (Fig. 1c). The significant difference in the density of haemocytes in the haemolymph collected from the heart and adductor muscle might be related to two factors: (i) frequency of haemocyte release from haematopoietic organs of invertebrates (Hoffmann, 1973), (ii) proximity of the haemolymph sampling site to widely speculated haematopoietic organ in the molluscs, the pericardium of the heart (Hartenstein, 2006). Interestingly, the THCs exhibited insignificant difference with the respective control values by 32 D of starvation although Fig. 6h reflects severely damaged pericardium. Hence, perceived homeostasis might also reflect exhaustion of the bivalve to meet its physiological demand under prolonged stress. However, the innate immune behaviour of the haemocytes of the starved bivalves depicts the commitment of the mollusc to maintain its immunological profile during nutritional stress. Although the phagocytic efficiency and ability to generate reactive nitrites exhibited hormesis during the 2 D, 4 D and 8 D of starvation, they regained homeostasis near to the control value by 32 D (Fig. 2d; Fig. 3). A significant correlation existed between the nitrite generated by the haemocytes from adductor muscle and the PI of the same haemocytes (Table 1). Phagocytosis is an energetically uphill process and requires substrates like glucose and adipose triglyceride lipase for its sustenance (Chandak et al., 2010). For the starving L. marginalis, its own tissue was the only source which it could have degenerated to yield the necessary ingredients of phagocytic energy. The mantle and foot muscle of the starved bivalve exhibited numerous vacuoles as signature of autolytic tissue degradation (Fig. 6b, h, and j). Notably, the adipocytes of the mantle tissue were severely vacuolated during starvation (Fig. 6b) which might have supplemented the bivalve with the energy. Nitric oxide, an innate immune molecule, is generated in haemocytes as a by-product during conversion of l-arginine to l-citrulline by the enzyme nitric oxide synthase (Bogdan, 2001). The starving bivalves were able to maintain a steady supply of nitrites till 32 D (Fig. 3)
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Table 1 Pearson’s correlation coefficient among total haemocyte count from heart haemolymph (TC-H), total haemocyte count from foot muscle haemolymph (TC-M), nitrite generation in heart haemocyte (NO-H), nitrite generation in foot muscle haemocyte (NO-M), phagocytic index of heart haemocyte (PI-H) and phagocytic index of foot muscle haemocyte (PI-M) of L. marginalis under starvation for a maximum time span of 32 D (n = 5).
D TC-H TC-M NO-H NO-M PI-H PI-M *
D
TC-H
TC-M
NO-H
NO-M
PI-H
PI-M
1.000 −0.220 −0.030 0.075 0.320 −0.240 0.350
1.000 0.650* −0.700 −0.810 −0.330 −0.720
1.000 −0.430 −0.580 −0.280 −0.390
1.000 0.710* −0.190 0.940*
1.000 −0.230 0.840*
1.000 −0.340
1.000
P < 0.05.
Table 2 The effects of treatment (control, starvation), time (D) and treatment-time interaction on L. marginalis under starvation for a maximum time span of 32 D. Dependent variable
Source of variation
df
Sum of squares
Mean square
F
P
THC of haemolymph from heart
Treatment Time Treatment × Time
1 6 6
160.772 264.094 261.211
160.772 44.016 43.535
322.293 88.237 87.273
0.000* 0.000* 0.000*
THC of haemolymph from muscle
Treatment Time Treatment × Time
1 6 6
20.650 96.176 71.582
20.650 16.029 11.930
534.851 415.168 309.002
0.000* 0.000* 0.000*
PI of haemocytes from heart
Treatment Time Treatment × Time
1 6 6
105.657 207,108.743 80,534.286
105.657 34,518.124 13,422.381
0.450 146.939 59.213
0.505 0.000* 0.000*
PI of haemocytes from muscle
Treatment Time Treatment × Time
1 6 6
488,057.500 660,663.571 12,290.743
488,057.500 110,110.595 2048.457
0.000 584.030 8.720
0.000* 0.000* 0.000*
NO generation in haemocytes from heart
Treatment Time Treatment × Time
1 6 6
4.740 2.393 3.060
4.740 0.399 0.510
559.179 47.050 60.171
0.000* 0.000* 0.000*
NO generation in haemocytes from muscle
Treatment Time Treatment × Time
1 6 6
0.009 0.308 0.013
0.009 0.051 0.002
11.212 65.704 2.716
0.001* 0.000* 0.022*
Total protein in haemolymph from heart Total protein in haemolymph from muscle
Treatment Time Treatment × Time Treatment Time Treatment × Time
1 3 3 1 3 3
1.940 2.754 2.717 2.093 3.099 3.363
1.940 0.918 0.906 2.093 1.033 1.121
162.768 76.997 75.973 105.477 52.060 56.486
0.000* 0.000* 0.000* 0.000* 0.000* 0.000*
Total protein in heart muscle
Treatment Time Treatment × Time
1 3 3
269.932 1031.496 1009.653
269.932 343.832 336.551
407.847 519.503 508.502
0.000* 0.000* 0.000*
Total protein in gill
Treatment Time Treatment × Time
1 3 3
16.609 128.929 129.692
16.609 42.976 43.231
16.084 41.618 41.864
0.000* 0.000* 0.000*
Total protein in digestive gland
Treatment Time Treatment × Time
1 3 3
732.650 1883.694 1877.290
732.650 627.898 625.763
94.161 80.698 80.424
0.000* 0.000* 0.000*
Activity of amylase at 10 min
Treatment Time Treatment × Time
1 3 3
0.016 0.014 0.015
0.016 0.005 0.005
12.588 3.701 3.742
0.001* 0.022* 0.021*
Activity of amylase at 20 min
Treatment Time Treatment × Time
1 3 3
0.000 0.006 0.007
0.000 0.002 0.002
0.082 1.667 1.991
0.777 0.194 0.135
Activity of amylase at 30 min
Treatment Time Treatment × Time
1 3 3
0.000 0.002 0.003
0.000 0.001 0.001
3.175 4.931 7.267
0.084 0.006* 0.001*
*
P < 0.05.
which imply that they had the necessary supply of l-arginine. Interestingly, the content of protein in the haemolymph, heart muscle, gill and digestive gland maintained homeostasis till 16 D and then increased significantly by 32 D (Fig. 4a and b). This could have only been made possible by the starving bivalve through autolytic degradation of tissue as observable in all the histopathological exhibits
(Fig. 6b, d, f, h, j, and l). The dietary stress had significant impact on the digestive tissue derived ␣-amylase activity only on 32 D (Fig. 5). The female gonadal tissue appeared to have suffered minimal visible damage probably due to its maximum resource reserve in the tissue matrix as well as in oocytes (Fig. 6m and n). However, depletion in the density of spermatids was observable in the male gonads
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Fig. 2. (a–c) Haemocytes of L. marginalis phagocytosing yeast spores (YS); (b) dynamics of phagocytic index (PI) of the haemocytes from heart and foot muscle of L. marginalis under starvation for a maximum time-span of 32 D (data presented as mean ± S.D.; n = 5; *P < 0.05).
Fig. 3. Dynamics of nitrite generation in the haemocytes from heart and foot muscle of L. marginalis under starvation for a maximum time span of 32 D (data presented as mean ± S.D.; n = 5; *P < 0.05).
(Fig. 6p). Besides, the shells of the starved bivalves were densely melanized (Fig. 1b) probably due to a standard defensive secretion by the phenoloxidase cascade (Allam and Raftos, 2015) of the molluscan mantle tissue and haemocytes under stress. Animals possess unique nutrient-sensing mechanisms (Efeyan et al., 2015) and during starvation they exercise an instinctive/adaptive measure of endogenous metabolism to replenish its
energy demand (McCue, 2010). Few animals including molluscs have a tendency to reduce their metabolic demand during starvation (Prosser, 1973). However, prolonged starvation affects some aspect of physiology and/or anatomy of the animals and thus cases like maintenance of glucose level in mollusc haemolymph till 30 days of starvation at the cost of degeneration of resource depot of mantle (Borges et al., 2004), alteration of nucleic acid, haema-
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Fig. 4. Dynamics of total protein (a) in the haemolymph from heart and foot muscle (b) in the heart muscle, gill and digestive gland of L. marginalis under starvation for a maximum time span of 32 D (data presented as mean ± S.D.; n = 5; *P < 0.05).
Fig. 5. Rates of generation of maltose by the activity of amylase in the digestive gland of L. marginalis under starvation for a maximum time span of 32 D (data presented as mean ± S.D.; n = 5; *P < 0.05).
tocrit content, digestive activity (Dimitriadis and Hondros, 1992; McCue, 2010; Moschovaki-Filippidou et al., 2013) are in report. The trait of sustaining costly immune expense is observable in the popular invertebrate model Drosophila melanogaster where Valtonen
et al. (2010) demonstrated that immunodeficient mutant flies with lower investments in immunological maintenance had greater life expectancy when put to starvation than wild type flies.
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Fig. 6. Histopathological profile of the vital organs of L. marginalis (a) the longitudinal section (LS) of the mantle of control bivalve with adipocytes (AD) and brush bordered epithelium; (b) LS of the mantle of 32 D starved bivalve exhibited infiltration by haemocytes (HC) and formation of granulomatous tissue (GT); (c) transverse section (TS) of the gill of the control bivalve exhibited gill filaments (GF), septum (SE) connecting the gill lamella and median water channel (WC); (d) TS of the gill of 32 D starved bivalve exhibited shrinkage in GF and SE, dilated WC and accumulation of HC in the inner lamella; (e) sagittal section (SS) of a labial palp of control bivalve exhibiting plicae (PL) lining the inner epithelium, and a smooth outer epithelium; (f) SS of a labial palp of 32 D starved bivalve exhibiting infiltration by HC in the subepithelial connective tissue (SCT); (g) TS of the heart of the control bivalve exhibiting ventricular chamber (VC), ventricular muscle (VM) and pericardium (P); (h) TS of the 32 D starved bivalve exhibiting dilated VC, distorted VM and vacuolated P; (i) TS of the foot muscle of control bivalve exhibiting lined with ciliary epithelium and densely arranged somatic muscle (SM) in array of vertical and horizontal layers; (j) TS of foot muscle of 32 D starved bivalve with loosely arranged SM; (k) TS of the intestine of control bivalve exhibited thick intestinal wall (IW); (l) TS of the intestine of 32 D starved bivalve exhibited thin IW with a dilated intestinal lumen (IL); (m) TS of the female gonadal tissue exhibiting oocytes (Oc) localized within each acini and several acini simultaneously connected to a single germinal duct (GD); (n) each Oc with vitelline layer (VL), perivitelline space (PVS); (o) TS of the male gonadal tissue of control bivalve with spermatids (ST); (p) TS of the male gonadal tissue of 32 D starved bivalve with few ST within the GD.
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Sustaining immunity during starvation in bivalve mollusc: A costly affair Elizabeth Mahapatra a , Dishari Dasgupta a , Navodipa Bhattacharya a , Suvrotoa Mitra a , Debakana Banerjee a , Soumita Goswami a , Nabanita Ghosh a , Avijit Dey a , Sudipta Chakraborty a,b,∗ a b
Parasitology and Immunology Laboratory, Department of Zoology, Maulana Azad College, 8, Rafi Ahmed Kidwai Road, Kolkata 700013, West Bengal, India Department of Zoology, Government General Degree College, Keshiary, Paschim Medinipur 721133, West Bengal, India
a r t i c l e
i n f o
Article history: Received 14 September 2016 Received in revised form 17 February 2017 Accepted 17 February 2017 Available online xxx Keywords: Starvation Bivalve Phagocytosis Nitric oxide Haemocytes
a b s t r a c t Complete or partial depletion of resource in a freshwater habitat is a common phenomenon. As a consequence, aquatic fauna including bivalve molluscs may be exposed to dietary stress on a seasonal basis. Haemocyte based innate immune profile of the freshwater mollusc Lamellidens marginalis (Bivalvia: Eulamellibranchiata) was evaluated under starvation induced stress for a maximum period of 32 days in a controlled laboratory condition. During starvation, the bivalve haemocytes maintained a homeostasis in phagocytic efficacy and nitric oxide generation ability with respect to the control. The mollusc maintained a significantly high protein content in its haemolymph and tissues under the nutritional stress with respect to the control. The dietary stress had no significant impact on the activity of digestive tissue derived ␣-amylase till sixteenth day but by 32 days the enzyme activity went down significantly. The histopathological profile revealed that the bivalve was adapted to maintain a steady immune profile by incurring degeneration of its own tissue structure. The total haemocyte count surged significantly till 16 days but differed insignificantly with respect to the control at 32 days implying probable haematopoietic exhaustion. The study reflects the instinctive urge of the bivalve to maintain immune physiology at heavy metabolic cost under nutrient limited condition. © 2017 Elsevier Ltd. All rights reserved.
1. Introduction Molluscs, including the bivalves, occupy an important position in the freshwater ecosystem of the tropics by stabilising it against natural as well as anthropogenic stresses (Dudgeon et al., 2006; Chakraborty et al., 2012; Fritts et al., 2015). The filter-feeding habits of molluscs, including bivalves, regulate the productivity and health of an aquatic environment (Officer et al., 1982; Newell, 2004). The sensitivity of the bivalves to the environmental stressors is manifested through the altered structural and functional attributes of their circulating haemocytes (Chakraborty et al., 2008; Calisi et al., 2008; Chakraborty and Ray, 2009; Martins and Costa, 2015; Matozzo, 2016) and tissues (Belcheva et al., 2011; Chakraborty et al., 2013; deOliveira et al., 2016). The circulating haemocytes of the molluscs constitute a vital component of its defence strategy
∗ Corresponding author at: Parasitology and Immunology Laboratory, Department of Zoology, Maulana Azad College, 8, Rafi Ahmed Kidwai Road, Kolkata 700013, West Bengal, India. E-mail address: [email protected] (S. Chakraborty).
and nutrient dynamics (Feng et al., 1977; Cheng, 1981). Phagocytosis of non-self particulates and generation of reactive nitrogen intermediates (RNIs) like nitric oxide are evolutionarily conserved strategies which are employed by the molluscan haemocytes to combat pathogenic insults (Araya et al., 2009; Chakraborty et al., 2009; Park et al., 2012). Studying the fluctuations of the molluscan haemocyte morphotypes with relation to diverse stressors seems important in order to perceive the environmental changes (Pamparinin et al., 2002). Bivalves thus have become a dependable model for environment biomonitoring (Sanders, 1993; FernándezTajes et al., 2011; Fritts et al., 2015) and their immunological profile represents the health of their resident environment (Sauvé et al., 2002; Girón-Pérez, 2010; Bhunia et al., 2016). Introduction of bivalve to aquasystems deprived of unionid fauna is a popular environment management strategy to incorporate trophic stability in oligotrophic environment (Patterson et al., 1999; Vaughn and Hakenkamp, 2001; Xu et al., 2008). Their inclusion in an existing population may improve its genetic diversity (Cope and Waller, 1995; Szumowski et al., 2012). The relocated bivalve population may thus be temporarily subjected to a con-
http://dx.doi.org/10.1016/j.tice.2017.02.005 0040-8166/© 2017 Elsevier Ltd. All rights reserved.
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dition of complete food deprivation due to the founder effect (Hoftyzer et al., 2008; Gilg et al., 2013) or the reinforced population may suffer from partial food deprivation due to augmented competition (Wacker and von Elert, 2002; Guernic et al., 2015). Partial or complete starvation may alter the biochemical composition of bivalves (Whyte et al., 1990), decrease their metabolic activity (Rodhouse and Gaffney, 1984) and change the haemocyte defence capacity (Butt et al., 2007). Lamellidens marginalis (Mollusca; Bivalvia) is a common bivalve mollusc of the freshwater aquasystems of eastern India including West Bengal (Fig. 1a). It is often used as a dietary supplement for poultry as well as human feed in rural West Bengal and adjoining states (Chakraborty et al., 2008; Madhyastha et al., 2010). It is a potential culturable aquacrop since pearls are naturally formed in the bivalve (Barik et al., 2004; Madhyastha et al., 2010). Its physiology has exhibited dose-responsiveness to diverse environmental pollutants like cadmium, chromium, arsenic (Das and Jana, 2004; Satyaparameshwar et al., 2006; Chakraborty et al., 2008, 2012, 2013) and stressors like oxygen and ammonia (Indira and Chetty, 1994; Das and Jana, 2003) which validates its prospective role in environment biomonitoring. However, the effect of starvation on the immunological profile of the haemocytes with relation to the morphological stability of tissue has not been investigated in details in molluscan model. The study was designed to understand the alterations in total count and selected immunological profiles of the haemocytes of L. marginalis subjected to dietary stress for a defined time span under controlled laboratory conditions. Histopathological study of selected organs of the starved molluscs would be helpful to characterise the effect of the stressor on its tissue structure. The data would be useful to analyse the impact of starvation induced stress on aquatic biota which would assist in formulation of a sustainable strategy of conservation of freshwater aquasystems.
2. Materials and methods
2.3. Enumeration of total haemocyte count (THC) Haemolymph was collected aseptically from the heart and posterior adductor muscle of the bivalves (Brousseau et al., 1999; Ahmad et al., 2011) in chilled glass vials. The THC was recorded separately from the haemolymph collected from the heart and posterior adductor muscle using Neubauer’s haemocytometer (Chakraborty et al., 2008). The enumeration of THC was carried out on haemolymph sampled from each of the 10 bivalves per experimental batch and the experiment was repeated for 5 times.
2.4. Enumeration of phagocytic index Cultured baker’s yeast (Saccharomyces cerevisiae) was killed by boiling for 1 h and the resulting cell suspension was washed three times in pH 7.4 TBS/Ca2+ (20 mM Trizma base, 77 mM NaCl, 10 mM CaCl2 ) by centrifugation at 650 × g for 10 min. The washed cells were then suspended at a concentration of 107 cells/ml in Grace’s Insect Medium (Himedia). The phagocytic efficiency of the haemocytes was examined by challenging them with yeast suspension in a ratio of about 1:10 in vitro over slide. To the adherent monolayer of haemocytes, 1 l of yeast (1 × 107 cells/ml) was added and incubated at 26 ◦ C in a humid chamber for 2 h. After incubation, the monolayer was washed with sterile snail saline (5 mM HEPES, 3.7 mM NaOH, 36 mM NaCl, 2 mM KCl, 2 mM MgCl2 ·2H2 O, 4 mM CaCl2 ·2H2 O; pH 7.8) (Adema et al., 1991), stained with Giemsa’s stain and observed under microscope (Axiostar Plus, Zeiss). A negative control for the assay was set using a known phagocytic inhibitor sodium azide (2%). More than 200 fields were examined for each slide and data was recorded to enumerate phagocytic index (PI), where PI = [(engulfed particles/phagocytic cells) × (phagocytic cells/total cells) × 100] (Elssner et al., 2004). The enumeration of PI was carried out on haemolymph sampled from each of the 10 bivalves per experimental batch and the experiment was repeated for 5 times.
2.1. Collection and maintenance of animals Fresh specimens of L. marginalis measuring 5 cm (±0.342) × 3.5 cm (±0.164) were sampled from the selected freshwater ponds of the district of 24-Parganas (South) of West Bengal in India. The collected animals (90 bivalves for each experiment) were transported to the laboratory and acclimated for 7 days in well-aerated tanks (90 cm × 90 cm × 60 cm) in batches of 15 per tank. They were provided with a feed of chopped lettuce leaves. The water of the tanks was replaced every 12 h to remove unutilized food and metabolic wastes. The average dissolved oxygen content and hardness of the water in the storage tanks were maintained at 14.1 mg/l and 457 mg/l respectively after Raut (1991). The water, soil sediments from the site of animal collection and bivalves from the sampling sites were tested periodically to trace heavy metal and metalloid contamination which yielded negative results.
2.5. Estimation of nitrite generation in haemocytes The estimation of the generation of NO was carried out on haemolymph pooled separately from the heart and posterior adductor muscle of the 10 bivalves per experimental batch. The generation of NO in the haemocyte was measured with Griess reagent modified after Green et al. (1982). The density of haemocytes was adjusted to 106 cells/ml and 1 ml of the haemocyte suspension was incubated with equal volume of Griess reagent (1% sulphanilamide, 0.1% naphthyl ethylenediamine dihydrochloride and 5% orthophosphoric acid) at 26 ◦ C for 30 min in a humid chamber. The absorbance was recorded in a spectrophotometer (Shimadzu 1800) at 550 nm against a standard blank. The generation of nitrite was determined using a standard curve of sodium nitrite. The generation of NO was expressed in terms of formation of nitrite as M/min/106 cells and the experiment was repeated for 5 times.
2.2. Exposure to stress 2.6. Preparation of tissue lysates Six separate batches comprising 10 bivalves per batch were subjected to starvation for a time span of 1, 2, 4, 8, 16 and 32 days (D) respectively. Each batch of the bivalves was maintained in wellaerated glass water tanks of 20 l capacity. One similar batch of bivalves was maintained with standard feed as control. The water of the tanks was replaced every 12 h to remove unutilized food and metabolic wastes. The experimental bivalves were monitored for possible visible health abnormalities and death. The experiments were repeated for 5 times (n = 5).
The tissue samples were dissected out of the heart, gill and digestive organ of the 10 bivalves per experimental batch and pooled separately before washing in chilled SSS. The tissue samples were weighed, homogenized in SSS and washed twice by centrifugation at 3000 rpm for 20 min at 4 ◦ C. The final pellet was resuspended with 1 ml of 0.1%. Triton X-100 and incubated under ice for 30 min. The suspensions were then centrifuged at 8000 rpm for 30 min at 4 ◦ C and the supernatants were collected and stored
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Fig. 1. (a) Control L. marginalis, (b) L. marginalis starved for 32 D exhibiting melanized shell and (c) dynamics of total haemocyte count in the haemolymph from heart and foot muscle of L. marginalis under starvation for a maximum time-span of 32 D (data presented as mean ± S.D.; n = 5; *P < 0.05).
in chilled vials at 4 ◦ C for not more than 1 h before being used for experimentation.
maltose was used to quantitate the optical densities and the kinetics of activity of ␣-amylase was expressed as rate of generation of maltose (M/min). The experiment was repeated for 5 times.
2.7. Estimation of total protein in haemolymph and tissue The estimation of the total protein of the haemolymph and tissue lysates was carried out spectrophotometrically (Shimadzu 1800) after Lowry et al. (1951) using a standard curve of bovine serum albumin. The experiment was repeated for 5 times. 2.8. Estimation of ˛-amylase activity in digestive tissue The kinetics of ␣-amylase (EC 3.2.1.1) activity was assayed by estimating the rate of generation of reducing sugars from starch by 3, 5-dinitrosalicyclic acid (DNS) method modified after Bertrand et al. (2004). In the process, 1 ml of the digestive tissue lysate was incubated with 1 ml of 1% starch solution (prepared in SSS) in 3 batches of test tubes. The reaction mixture in each of the three batches of test tubes was incubated in a humid chamber at 26 ◦ C for 10 min, 20 min and 30 min respectively. A blank consisting of 1 ml of 1% starch solution and 1 ml of SSS was incubated in identical conditions. The reaction was terminated by adding 2 ml of DNS reagent in each test tube and then incubating the tubes in a boiling water bath for 10 min. The test tubes were allowed to cool and the absorbance of the final reaction mixtures was spectrophotometrically (Shimadzu 1800) measured at 540 nm. A standard curve of
2.9. Histopathology of vital tissues The tissue samples were collected from the mantle, gill, labial palp, heart, foot muscle, gut and gonad of the control and 32 D starved animals. After autopsy, the tissue samples were washed in SSS and further processed for standard histological preparations after Chakraborty et al. (2010). The tissue sections were stained with haematoxylin-eosin, mounted in DPX, studied under microscope (Axiostar Plus, Zeiss). The documentation of tissue dimensions was carried out with an oculometer calibrated against a stage micrometre (Erma, Japan) after Carballal et al. (1997). 2.10. Statistical analysis The mean values of the experimental control and starved animals were compared using factorial Analysis of Variance (ANOVA). Pearson’s correlation coefficient was calculated to analyse relationship amongst the observed parameters of the bivalve along the entire life-span of starvation induced stress. SPSS Statistics versions 16 software was used for processing of the data (p < 0.05) and the data were expressed as mean ± standard deviation (S.D.).
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3. Results
3.6. Histopathology
3.1. THC
All histopathological exhibits were sampled from the control and 32 D starved bivalves. The mantle of the starved bivalve revealed intense haemocyte infiltration, development of granulomatous tissue and formation of vacuole in the adipocytes (Fig. 6a and b). The starved gill and labial palp of the bivalve also exhibited accumulation of hyperchromatic haemocytes within the inner lamellar space and subepithelial connective tissue. The central water channel of the starved gill was dilated at places and the gill filaments and the ciliated epithelium of the labial palp appeared to have suffered shrinkage (Fig. 6c–f). The starved bivalve exhibited intense inflammatory tissue damage in ventricular and the pericardial muscle of the heart (Fig. 6e and f), loss of compactness in the somatic muscle of the foot due to development of numerous vacuoles (Fig. 6g and h) and thinning of the intestinal wall with dilation of its lumen (Fig. 6i and j). The female gonadal tissue of the bivalve seemed least affected by starvation and exhibited normal tissue architecture for the acini and germinal duct (Fig. 6k). The oocytes appeared to be healthy with distinct vitelline layer and perivitelline space (Fig. 6l). The male gonadal tissue of the starved bivalve apparently maintained tissue composure although the density of the spermatids was visibly low with respect to the control animals (Fig. 6o and p).
The THC in the haemolymph collected from the heart of the bivalves exhibited a significant increase by 4 D of starvation as 10.34 × 106 ± 1.005 cells/ml with respect to the control value of 0.4 × 106 ± 0.134 cells/ml. Eventually the THC attained a value as 0.33 × 106 ± 0.324 cells/ml by 32 D without any significant difference with respect to the control (Fig. 1c). The THC in the haemolymph collected from the posterior adductor muscle of the bivalves exhibited identical pattern with the progression of starvation induced stress till 32 D with the highest count recorded on 4 D as 1.4 × 106 ± 0.08 cells/ml with respect to the control value of 0.271 × 106 ± 0.012 cells/ml (Fig. 1c; Table 2). 3.2. Phagocytic index The haemocytes of the bivalve exhibited varying phagocytic ability under exposure of starvation induced stress (Fig. 2a–c). The PI of the haemocytes collected from haemolymph of the heart of the bivalves exhibited no significant alteration with respect to the control value of 145 ± 12.7 with the only exception being on 2 D of starvation when the PI value was enumerated as high as 316 ± 19.432 (Fig. 2d). On the contrary, on 2 D of starvation, the PI value of the haemocytes collected from the posterior adductor muscle of the bivalve exhibited a significant drop to 113 ± 15.318 with respect to the control value of 467 ± 13.675. However, the PI value exhibited no significant difference by 32 D of starvation with respect to the control (Fig. 2d; Table 2). 3.3. Nitrite generation in haemocytes The generation of nitrite by the haemocytes collected from haemolymph of the heart of the bivalves remained significantly lower from 2 D till 8 D of starvation with respect to the control. However, the generation of nitrite attained insignificant deviation with respect to the control by 32 D of starvation (Fig. 3). The generation of nitrite by the haemocytes collected from haemolymph of the posterior adductor muscle of the bivalves exhibited identical pattern of fluctuation with respect to the control values. However, the generation of nitrite attained insignificant deviation with respect to the control by 16 D of starvation (Fig. 3; Table 2). 3.4. Total protein in haemolymph and tissue The content of total protein in the haemolymph from heart and posterior adductor muscle of the bivalve exhibited significant increase by 16 D and 32 D of starvation with respect to the control (Fig. 4a). The protein content of the heart muscle, gill and digestive gland tissue also increased in identical pattern by 32 D of starvation as 31.25 ± 0.832 mg/gm, 10 ± 2.041 mg/gm and 33.3 ± 3.321 mg/gm with respect to the control values of 8.670 ± 0.613 mg/gm, 2.560 ± 1.102 mg/gm and 1.037 ± 0.112 mg/gm respectively (Fig. 4b; Table 2). 3.5. Amylase activity in digestive tissue The kinetics of generation of maltose from starch due to amylase activity by the digestive tissue lysate from the bivalve exhibited a significant reduction by 32 D of starvation as 0.033 ± 0.002 M/min and 0.01 ± 0.006 M/min at 10 and 30 min with respect to the control values of 0.122 ± 0.002 M/min and 0.031 ± 0.004 M/min at 10 and 30 min respectively (Fig. 5; Table 2).
4. Discussion The study depicts the primal instinct of the bivalve mollusc to attain and maintain physiological homeostasis, only at a cost. The surge in the THC with the progression of starvation till eighth day probably reflects the innate response of the bivalve to adjust with the stress (Fig. 1c). The significant difference in the density of haemocytes in the haemolymph collected from the heart and adductor muscle might be related to two factors: (i) frequency of haemocyte release from haematopoietic organs of invertebrates (Hoffmann, 1973), (ii) proximity of the haemolymph sampling site to widely speculated haematopoietic organ in the molluscs, the pericardium of the heart (Hartenstein, 2006). Interestingly, the THCs exhibited insignificant difference with the respective control values by 32 D of starvation although Fig. 6h reflects severely damaged pericardium. Hence, perceived homeostasis might also reflect exhaustion of the bivalve to meet its physiological demand under prolonged stress. However, the innate immune behaviour of the haemocytes of the starved bivalves depicts the commitment of the mollusc to maintain its immunological profile during nutritional stress. Although the phagocytic efficiency and ability to generate reactive nitrites exhibited hormesis during the 2 D, 4 D and 8 D of starvation, they regained homeostasis near to the control value by 32 D (Fig. 2d; Fig. 3). A significant correlation existed between the nitrite generated by the haemocytes from adductor muscle and the PI of the same haemocytes (Table 1). Phagocytosis is an energetically uphill process and requires substrates like glucose and adipose triglyceride lipase for its sustenance (Chandak et al., 2010). For the starving L. marginalis, its own tissue was the only source which it could have degenerated to yield the necessary ingredients of phagocytic energy. The mantle and foot muscle of the starved bivalve exhibited numerous vacuoles as signature of autolytic tissue degradation (Fig. 6b, h, and j). Notably, the adipocytes of the mantle tissue were severely vacuolated during starvation (Fig. 6b) which might have supplemented the bivalve with the energy. Nitric oxide, an innate immune molecule, is generated in haemocytes as a by-product during conversion of l-arginine to l-citrulline by the enzyme nitric oxide synthase (Bogdan, 2001). The starving bivalves were able to maintain a steady supply of nitrites till 32 D (Fig. 3)
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Table 1 Pearson’s correlation coefficient among total haemocyte count from heart haemolymph (TC-H), total haemocyte count from foot muscle haemolymph (TC-M), nitrite generation in heart haemocyte (NO-H), nitrite generation in foot muscle haemocyte (NO-M), phagocytic index of heart haemocyte (PI-H) and phagocytic index of foot muscle haemocyte (PI-M) of L. marginalis under starvation for a maximum time span of 32 D (n = 5).
D TC-H TC-M NO-H NO-M PI-H PI-M *
D
TC-H
TC-M
NO-H
NO-M
PI-H
PI-M
1.000 −0.220 −0.030 0.075 0.320 −0.240 0.350
1.000 0.650* −0.700 −0.810 −0.330 −0.720
1.000 −0.430 −0.580 −0.280 −0.390
1.000 0.710* −0.190 0.940*
1.000 −0.230 0.840*
1.000 −0.340
1.000
P < 0.05.
Table 2 The effects of treatment (control, starvation), time (D) and treatment-time interaction on L. marginalis under starvation for a maximum time span of 32 D. Dependent variable
Source of variation
df
Sum of squares
Mean square
F
P
THC of haemolymph from heart
Treatment Time Treatment × Time
1 6 6
160.772 264.094 261.211
160.772 44.016 43.535
322.293 88.237 87.273
0.000* 0.000* 0.000*
THC of haemolymph from muscle
Treatment Time Treatment × Time
1 6 6
20.650 96.176 71.582
20.650 16.029 11.930
534.851 415.168 309.002
0.000* 0.000* 0.000*
PI of haemocytes from heart
Treatment Time Treatment × Time
1 6 6
105.657 207,108.743 80,534.286
105.657 34,518.124 13,422.381
0.450 146.939 59.213
0.505 0.000* 0.000*
PI of haemocytes from muscle
Treatment Time Treatment × Time
1 6 6
488,057.500 660,663.571 12,290.743
488,057.500 110,110.595 2048.457
0.000 584.030 8.720
0.000* 0.000* 0.000*
NO generation in haemocytes from heart
Treatment Time Treatment × Time
1 6 6
4.740 2.393 3.060
4.740 0.399 0.510
559.179 47.050 60.171
0.000* 0.000* 0.000*
NO generation in haemocytes from muscle
Treatment Time Treatment × Time
1 6 6
0.009 0.308 0.013
0.009 0.051 0.002
11.212 65.704 2.716
0.001* 0.000* 0.022*
Total protein in haemolymph from heart Total protein in haemolymph from muscle
Treatment Time Treatment × Time Treatment Time Treatment × Time
1 3 3 1 3 3
1.940 2.754 2.717 2.093 3.099 3.363
1.940 0.918 0.906 2.093 1.033 1.121
162.768 76.997 75.973 105.477 52.060 56.486
0.000* 0.000* 0.000* 0.000* 0.000* 0.000*
Total protein in heart muscle
Treatment Time Treatment × Time
1 3 3
269.932 1031.496 1009.653
269.932 343.832 336.551
407.847 519.503 508.502
0.000* 0.000* 0.000*
Total protein in gill
Treatment Time Treatment × Time
1 3 3
16.609 128.929 129.692
16.609 42.976 43.231
16.084 41.618 41.864
0.000* 0.000* 0.000*
Total protein in digestive gland
Treatment Time Treatment × Time
1 3 3
732.650 1883.694 1877.290
732.650 627.898 625.763
94.161 80.698 80.424
0.000* 0.000* 0.000*
Activity of amylase at 10 min
Treatment Time Treatment × Time
1 3 3
0.016 0.014 0.015
0.016 0.005 0.005
12.588 3.701 3.742
0.001* 0.022* 0.021*
Activity of amylase at 20 min
Treatment Time Treatment × Time
1 3 3
0.000 0.006 0.007
0.000 0.002 0.002
0.082 1.667 1.991
0.777 0.194 0.135
Activity of amylase at 30 min
Treatment Time Treatment × Time
1 3 3
0.000 0.002 0.003
0.000 0.001 0.001
3.175 4.931 7.267
0.084 0.006* 0.001*
*
P < 0.05.
which imply that they had the necessary supply of l-arginine. Interestingly, the content of protein in the haemolymph, heart muscle, gill and digestive gland maintained homeostasis till 16 D and then increased significantly by 32 D (Fig. 4a and b). This could have only been made possible by the starving bivalve through autolytic degradation of tissue as observable in all the histopathological exhibits
(Fig. 6b, d, f, h, j, and l). The dietary stress had significant impact on the digestive tissue derived ␣-amylase activity only on 32 D (Fig. 5). The female gonadal tissue appeared to have suffered minimal visible damage probably due to its maximum resource reserve in the tissue matrix as well as in oocytes (Fig. 6m and n). However, depletion in the density of spermatids was observable in the male gonads
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Fig. 2. (a–c) Haemocytes of L. marginalis phagocytosing yeast spores (YS); (b) dynamics of phagocytic index (PI) of the haemocytes from heart and foot muscle of L. marginalis under starvation for a maximum time-span of 32 D (data presented as mean ± S.D.; n = 5; *P < 0.05).
Fig. 3. Dynamics of nitrite generation in the haemocytes from heart and foot muscle of L. marginalis under starvation for a maximum time span of 32 D (data presented as mean ± S.D.; n = 5; *P < 0.05).
(Fig. 6p). Besides, the shells of the starved bivalves were densely melanized (Fig. 1b) probably due to a standard defensive secretion by the phenoloxidase cascade (Allam and Raftos, 2015) of the molluscan mantle tissue and haemocytes under stress. Animals possess unique nutrient-sensing mechanisms (Efeyan et al., 2015) and during starvation they exercise an instinctive/adaptive measure of endogenous metabolism to replenish its
energy demand (McCue, 2010). Few animals including molluscs have a tendency to reduce their metabolic demand during starvation (Prosser, 1973). However, prolonged starvation affects some aspect of physiology and/or anatomy of the animals and thus cases like maintenance of glucose level in mollusc haemolymph till 30 days of starvation at the cost of degeneration of resource depot of mantle (Borges et al., 2004), alteration of nucleic acid, haema-
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Fig. 4. Dynamics of total protein (a) in the haemolymph from heart and foot muscle (b) in the heart muscle, gill and digestive gland of L. marginalis under starvation for a maximum time span of 32 D (data presented as mean ± S.D.; n = 5; *P < 0.05).
Fig. 5. Rates of generation of maltose by the activity of amylase in the digestive gland of L. marginalis under starvation for a maximum time span of 32 D (data presented as mean ± S.D.; n = 5; *P < 0.05).
tocrit content, digestive activity (Dimitriadis and Hondros, 1992; McCue, 2010; Moschovaki-Filippidou et al., 2013) are in report. The trait of sustaining costly immune expense is observable in the popular invertebrate model Drosophila melanogaster where Valtonen
et al. (2010) demonstrated that immunodeficient mutant flies with lower investments in immunological maintenance had greater life expectancy when put to starvation than wild type flies.
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Fig. 6. Histopathological profile of the vital organs of L. marginalis (a) the longitudinal section (LS) of the mantle of control bivalve with adipocytes (AD) and brush bordered epithelium; (b) LS of the mantle of 32 D starved bivalve exhibited infiltration by haemocytes (HC) and formation of granulomatous tissue (GT); (c) transverse section (TS) of the gill of the control bivalve exhibited gill filaments (GF), septum (SE) connecting the gill lamella and median water channel (WC); (d) TS of the gill of 32 D starved bivalve exhibited shrinkage in GF and SE, dilated WC and accumulation of HC in the inner lamella; (e) sagittal section (SS) of a labial palp of control bivalve exhibiting plicae (PL) lining the inner epithelium, and a smooth outer epithelium; (f) SS of a labial palp of 32 D starved bivalve exhibiting infiltration by HC in the subepithelial connective tissue (SCT); (g) TS of the heart of the control bivalve exhibiting ventricular chamber (VC), ventricular muscle (VM) and pericardium (P); (h) TS of the 32 D starved bivalve exhibiting dilated VC, distorted VM and vacuolated P; (i) TS of the foot muscle of control bivalve exhibiting lined with ciliary epithelium and densely arranged somatic muscle (SM) in array of vertical and horizontal layers; (j) TS of foot muscle of 32 D starved bivalve with loosely arranged SM; (k) TS of the intestine of control bivalve exhibited thick intestinal wall (IW); (l) TS of the intestine of 32 D starved bivalve exhibited thin IW with a dilated intestinal lumen (IL); (m) TS of the female gonadal tissue exhibiting oocytes (Oc) localized within each acini and several acini simultaneously connected to a single germinal duct (GD); (n) each Oc with vitelline layer (VL), perivitelline space (PVS); (o) TS of the male gonadal tissue of control bivalve with spermatids (ST); (p) TS of the male gonadal tissue of 32 D starved bivalve with few ST within the GD.
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