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Reproductive phase dependent variation in lungassociated immune system (LAIS) and expression of melatonin receptors (Mel1a and Mel1b) in the lung of the Jungle-Bush Quail (Perdicula asiatica) Can. J. Zool. Downloaded from www.nrcresearchpress.com by MICHIGAN STATE UNIV on 01/16/17 For personal use only.

R.K. Kharwar and C. Haldar

Abstract: The present study was performed to assess the variation of the lung-associated immune system (LAIS) in the Jungle-Bush Quail (Perdicula asiatica (Latham, 1790)) during two different reproductive phases when differences in the circulatory level of hormones (melatonin and gonadal steroid) and environmental conditions were maximum. We noted high significant variation in size and number of bronchus-associated lymphoid tissue (BALT) nodules, as well as in the size and number of non-BALT nodules, during the reproductively inactive phase (RIP; December) compared with the active phase (RAP; June). We also noted high significant variation in the percent stimulation ratio of lung lymphocyte, as well as in the concentrations of plasma melatonin and melatonin receptors, during RIP compared with RAP. Testosterone level and number of macrophages in lungs were high during RAP. Thus, we suggest that the LAIS had reproductive phase dependent variation, which could be due to (i) variation in environmental factors (photoperiod, temperature, and humidity) and (ii) circulatory level of hormones (melatonin and testosterone). Because of the importance of melatonin in avian immune regulation, we assess and document the expression of melatonin receptor types Mel1a and Mel1b in the avian lung, which suggest that the lung is a target organ for melatonin and that melatonin is an immunomodulator for lung-associated immunity in birds. Re´sume´ : Notre e´tude vise a` e´valuer la variation du syste`me immunitaire pulmonaire (LAIS) chez la perdicule roussegorge (Perdicula asiatica (Latham, 1790)) pendant deux phases reproductives distinctes durant lesquelles les diffe´rences dans les niveaux des hormones en circulation (me´latonine et ste´roı¨des gonadiques) et dans les conditions du milieu sont maximales. Nous notons de plus fortes variations significatives dans la taille et le nombre de nodules de tissu lymphoı¨de associe´ aux bronches (BALT), ainsi que dans la taille et le nombre des nodules non de BALT, durant la pe´riode sans reproduction (RIP, de´cembre) que durant la phase active de la reproduction (RAP, juin). Il y aussi une forte variation du rapport % de stimulation des lymphocytes pulmonaires, des concentrations de me´latonine plasmatique et des re´cepteurs de la me´latonine durant la RIP par comparaison avec la RAP. Le niveau de testoste´rone et le nombre de macrophages dans les poumons sont e´leve´s durant la RAP. Nous pouvons donc supposer que le LAIS connaıˆt une variation de´pendante de la phase reproductive qui pourrait eˆtre due (i) a` la variation des facteurs environnementaux (photope´riode, tempe´rature et hu` cause de l’importance de la me´latomidite´) et (ii) au niveau des hormones en circulation (me´latonine et testoste´rone). A nine dans la re´gulation immunitaire chez les oiseaux, nous e´valuons et de´crivons l’expression des re´cepteurs de me´latonine de types Mel1a et Mel1b dans le poumon ce qui nous laisse croire que le poumon est un organe cible de la me´latonine et que la me´latonine sert d’immunomodulateur dans l’immunite´ associe´e au poumon chez les oiseaux. [Traduit par la Re´daction]

Introduction Avian species have been studied mostly for the reproductive physiology, behavior, metabolism (Linden and Møller 1989; Gustafsson et al. 1994; Bentley et al. 1998; Budki et al. 2009), and the immune system (Lee 2006; Martin et al. 2006; Singh and Haldar 2007; Davison et al. 2008). The study of the immune system of wild birds by ecologists is Received 13 March 2010. Accepted 12 October 2010. Published on the NRC Research Press Web site at cjz.nrc.ca on 11 December 2010. R.K. Kharwar and C. Haldar.1 Pineal Research Laboratory, Department of Zoology, Banaras Hindu University, Varanasi – 221 005, India. 1Corresponding

author (e-mail: [email protected]).

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not yet as rigorous as the study of the immune system of chickens by immunologists. Furthermore, immune function in the lung of wild birds has not been studied. Recent epidemics such as avian influenza or bird flu have dragged attention of researchers towards the study of the immune status of birds. This is because the avian respiratory system is in direct contact with the external environment, and hence, the lung becomes a major target organ for numerous threatening agents in the air that induce several respiratory diseases. These diseases have a major impact on poultry production throughout the world, often bringing economic loss. Pathogens invade the lung and from there migrate to other regions of body. To overcome such problems, the respiratory system is equipped with a well-organized immune system. The lung-associated immune system (LAIS) has been studied in various mammals (Delventhal et al. 1992;

doi:10.1139/Z10-091

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Kharwar and Haldar

Sato et al. 2000), as well as in domestic poultry such as chickens (Bienenstock et al. 1973a, 1973b) and turkeys (Fagerland and Arp 1990). The Indian Jungle-Bush Quail (Perdicula asiatica (Latham, 1790)) is a seasonal breeder. Its breeding season extends from late March to July. Substantial work has been done on general immunity (Singh and Haldar 2005, 2007) and reproductive aspect (Haldar and Ghosh 1990; Sudhakumari et al. 2001) of this species, but no work has been carried out on LAIS of this species. Major components of avian LAIS are bronchus-associated lymphoid tissue (BALT) nodules and non-BALT nodules, which both consist of lymphocyte (T cells and B cells) aggregates. BALT and non-BALT nodules play a crucial role in the development of local immune response to inhaled antigens (Bienenstock et al. 1973b; Fagerland and Arp 1990; Jeurissen et al. 1994; Reese et al. 2006). Non-BALT nodules found adjacent to blood vessels in P. asiatica (Kharwar and Haldar 2010) suggest an opportunity for lymphocytes and humoral products to enter directly into the vascular system, which might conceivably provide extra local protection for seasonally occurring stress. Melatonin is known for its effect on general immunity of birds (Skwarlo-Sonta 1999; Singh and Haldar 2007), as well as for being an antioxidant (Albarra´n et al. 2001; Rodriguez et al. 2004). Melatonin utilizes its various receptors on target tissues for different actions. It has also been established that two cloned membrane melatonin receptors, i.e., Mel1a and Mel1b, exist on immune organs and on immune cells of vertebrates (Calvo et al. 1995; Carrillo-Vico et al. 2003). In nonmammalian species, another membrane receptor (Mel1c) has frequently been demonstrated, but because of the lack of specific commercial antibody for use against this receptor, its function has not been established. However, no single study exists that shows variation in LAIS for avian species in general, or for seasonally breeding birds in particular, when it was established that (i) environmental factors (photoperiod, temperature, humidity, and food availability), social factors, and stress influence bird physiology on the one hand and (ii) the circulatory level of hormones (gonadal, adrenal steroid, and melatonin levels) affects the general immune status (Singh and Haldar 2005) on the other hand. In the wild, P. asiatica encounters more respiratory stress than domestic poultry such as chickens and turkeys. Therefore, we thought it worthwhile to study in detail the variation in LAIS of a tropical seasonal breeder (P. asiatica) during the active (June) and inactive (January) reproductive phases. Because of the importance of melatonin in avian immunomodulation (Singh and Haldar 2007), we paid special attention to the expression of melatonin receptors Mel1a and Mel1b in the lungs of P. asiatica to suggest that LAIS could be the target of melatonin.

Materials and methods Husbandry Experiments were conducted on healthy adult male P. asiatica (body mass ~45 g). This species is sexually dimorphic, with males generally being smaller in size than females. In addition to the sexual dimorphism, females have a dark patch on the ventral side of the neck. For the present study, males were collected during the reproductively active

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period (RAP; first week of June) and during the reproductively inactive period (RIP; first week of January) near Varanasi (25818’N, 8381’E) and acclimatized to laboratory conditions for 2 weeks in an aviary exposed to ambient environmental conditions (RAP, June: approximately 14 h light : 10 h dark photoperiod, with temperatures 37 ± 5 8C (maximum) and 26 ± 5 8C (minimum) and approximately 65% humidity; RIP, January: approximately 11 h light : 13 h dark photoperiod, with temperatures 15 ± 5 8C (maximum) and 6 ± 3 8C (miminum) and approximately 90% humidity). They were fed seeds of the pearl millet (Pennisetum glaucum (L.) R. Br.), as well as other seasonal grains, and provided water ad libitum (Singh and Haldar 2007). All experiments were performed in accordance with institutional practice and within the framework of the revised Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) Act of 2007 of Government of India. Experimental protocol Twenty-eight P. asiatica captured during RAP were divided as follows: 7 birds were used for histological study, 7 birds were used for immunohistochemical study, 7 birds were used for percent stimulation ratio (%SR) of lung lymphocytes, and 7 birds were used for Western blot analysis of lung protein for Mel1a and Mel1b receptors. Twenty-eight birds captured during RIP were also divided as above. Histological study To perform histological observation of the lung and to study BALT and non-BALT nodular changes, seven adult male P. asiatica (mass ~45 g) captured during RAP and RIP were euthanized under complete anesthesia with Nembutal (sodium pentobarbital) during the late-evening hours of 1930–2030 (approximately 3 h after sunset). Within a very short period of time, the lungs were saturated with Bouin’s fluid in situ and dissected out of the birds. The lungs were then cleaned and fixed again by immersion overnight in Bouin’s fluid in preparation for routine histology. After fixation, tissues were dehydrated and embedded in paraffin. Transverse sections of entire lung (~1 cm  0.75 cm) cut at 6 mm thickness were stained with Harris hematoxylin and eosin (1% alcoholic). The size of BALT and non-BALT nodules in lung tissue was determined histologically with the aid of a Filar ocular micrometer (WEBCON, Varanasi, India). Ten sections of the entire lung from each bird during RAP and RIP were randomly selected for morphometric analysis of 20 BALT and 20 nonBALT nodules to present the changes observed during the two reproductive phases. Immunohistochemistry Melatonin receptor types (Mel1a and Mel1b) have been reported in the lungs of P. asiatica (Kharwar and Haldar 2010). To record the variation during RIP and RAP, immunohistochemistry was performed. After Nembutal anesthesia, seven birds were perfused with 4% paraformaldehyde (PFA) and the entire lung was dissected out and kept overnight in 4% PFA. After dehydration, paraffin blocks were prepared and 6 mm thick transverse sections were cut and mounted on 1% gelatin coated slides and deparaffinized. Endogenous peroxidase activity was blocked by H2O2 in 80% methanol Published by NRC Research Press

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for 20 min at room temperature. Sections were washed three times with phosphate-buffered saline (PBS) and preincubated with 3% blocking serum in PBS for 40 min. Sections were then incubated overnight at 4 8C with primary antibodies Mel1a (MEL-1A-R (R-18): sc-13186) and Mel1b (MEL-1B-R (T-18): sc-13177), which are affinity-purified goat polyclonal antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, California, USA; dilution 1:200). Sections were washed three times in PBS and were incubated with biotinylated secondary antibodies (Vectastain ABC Universal kit, PK-6200; Vector Laboratories, Burlinghame, California, USA; dilution 1 : 10 000). Sections were washed with PBS and a preformed ABC reagent was conjugated to the free biotin of the secondary antibody. The antigens were visualized using the peroxidase substrate 3,3-diaminobenzidine (DAB) (Savaskan et al. 2002; Kharwar and Haldar 2010). %SR of lymphocytes isolated from lung tissue To present the LAIS in terms of %SR, lungs were dissected out and cleaned of adherent tissues, washed with PBS, and minced into small pieces with scissors over a sterile watch glass. The pieces of lung were then passed through a steel strainer (400 mm mesh size) and the cell suspension was collected in a sterile centrifuge tube and washed twice with RPMI-1640. The cell viability was checked with the 1.0% Trypan blue exclusion method. In the cell suspension, the red cells were lysed with cold ammonium chloride TRIS (Tris(hydroxymethyl)aminomethane) buffer (BDH Chemicals, Poole, Dorset, UK), which consists of 0.5% TRIS buffer and 0.84% NH4Cl mixed in a 1:10 ratio and adjusted to pH 7.2. Single cell suspension of isolated lung lymphocytes was adjusted to 1  10 6 cells/mL in supplemented RPMI-1640, containing sodium bicarbonate, antibiotics (penicillin 100 IU/mL, streptomycin 100 mg/mL, gentamycin 100 mg/mL), and 10% fetal calf serum (Sigma–Adrich, St. Louis, Missouri, USA). For the study of blastogenic response, the cell suspension was divided into aliquots of 2 mL each having 1  106 cells/mL. The control plates were cultured in the absence of mitogen, whereas the test cultures were stimulated with mitogen Concanavalin A (5 mg/mL). The plates were incubated at 41 8C under 5% CO2 in a HERAcell CO2 incubator (Kendro Laboratory Products (India) Pvt. Ltd., New Delhi, India) for 72 h. Eighteen hours before harvesting, 1 mCi of tritiated thymidine [3H] (Bhabha Atomic Research Centre (BARC), Mumbai, India; specific activity 8.9 Ci/mM, where 1 mM is 1 mmol/L) was added to each culture plate. Culture plates were harvested after 72 h of incubation. Blastogenic response was measured in terms of [3H] thymidine uptake against stimulation by Concanavalin A of the lung lymphocytes (Pauly and Sokal 1972). The data were presented as %SR = (counts per minute with Concanavalin A / counts per minute without Concanavalin A)  100. Hormonal analysis Before perfusion with 4% paraformaldehyde, blood was collected directly from the heart within a very short period of time in a heparinized syringe, centrifuged, and plasma was stored in –20 8C for the RIA of testosterone. For RIA of melatonin, blood from the pectoral vein was collected during the night hour of 2300 prior to the day when the

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P. asiatica was euthanized; after centrifugation, the plasma was stored in –20 8C. For hormonal analysis of plasma melatonin, a radioimmunoassay was done following the modified method of Rollag and Niswender (1976). The RIA kit from Radioassay Systems Laboratories, Inc. (Carson, California, USA) was used for plasma testosterone assay. The validation of RIA was performed and described earlier (Sudhakumari et al. 2001). The sensitivity for melatonin RIA was 18–20 pg/mL and for testosterone RIA was 6 pg/mL. The intra- and interassay variations for melatonin were 9% and 15%, respectively, and for testosterone were 4.5% and 5.6%, respectively. The recoveries of testosterone and melatonin RIA were 95% and 92%, respectively. Western blot analysis To ensure the expression of Mel1a and Mel1b from lung protein at the translational level during the two reproductive phases, a piece of lung was removed and lysed in RIPA buffer (consisting of 1% (v/v) Igel CA-630, 0.5% (w/v) sodium deoxycholate, 0.1% (m/v) sodium dodecyl sulfate (SDS) in phosphate-buffered solution (PBS) containing aprotonin and sodium orthovanadate). Aliquots containing 100 mg of protein (Roy et al. 2001; Kharwar and Haldar 2010) were resolved by 12% (m/v) SDS–PAGE along with Spectra Multicolor Broad Range Protein Marker (SM1841) to determine the immunoreactive bands and was followed by electrotransfer to a PVDF hybond membrane (Amersham, Buckinghamshire, UK). Immunodetection was carried out using melatonin receptor antibodies Mel1a (R-18: sc-13186), and Mel1b (T-18: sc-13177) (Santa Cruz Biotechnology, Inc., Santa Cruz, California, USA; dilution 1:200) followed by horseradish peroxidase conjugated secondary antibody (antigoat donkey IgG), which was detected using chemiluminescence (ECL) system (Amersham, Buckinghamshire, UK). Each band was quantified with the aid of the Scion Image analysis software (http://www.scioncorp.com; accessed 22 August 2007). The ratio of density was calculated after normalization with b-actin and was expressed as percent control value (Treeck et al. 2006; Kharwar and Haldar 2010). Statistical analysis The data for the size of BALT and non-BALT nodules, for hormonal level, and for Western blot analysis during RAP and RIP were analyzed with a two-way ANOVA followed by Student–Newman–Keuls’ multiple range test and Student’s t test wherever applicable. Results are expressed as mean ± SE. The differences were considered significant at p < 0.05 and highly significant at p < 0.01 (Bruning and Knitz 1977). Microsoft Excel was used for statistical calculations and data presentation.

Results Histological observation The BALT nodule can be differentiated from non-BALT nodules by the aggregation of lymphocytes located on the junction of primary bronchus and secondary bronchi (Figs. 1a, 1c), whereas non-BALT nodules had aggregation of lymphocytes on other regions of the lungs (Figs. 1b, 1d). Interestingly, the size and number of BALT and non-BALT Published by NRC Research Press

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Fig. 1. Transverse section (hematoxylin and eosin stain) of lung tissue of male Jungle-Bush Quail (Perdicula asiatica) showing (a) BALT nodule (bn) during the reproductively inactive period (RIP), (b) non-BALT nodule (nbn) during RIP, (c) BALT nodule (bn) during the reproductively active period (RAP), and (d) non-BALT nodule (nbn) during RAP.

nodules varied with the different reproductive phases. Numerous BALT nodules were significantly larger in size (p < 0.01) in lung tissues during RIP than during RAP (Figs. 1a–1d, 3a). A number of lymphocytes and macrophages were observed either in groups or in free form throughout the lung tissue. We found a larger number of macrophages in BALT nodules during RAP than during RIP (Figs. 2a, 2b). Morphometric analysis of BALT and non-BALT nodules suggest that non-BALT nodules were significantly larger in size (p < 0.01) than BALT nodules during RAP, whereas no significant difference was observed in size of both the nodule types during RIP (Fig. 3a). Immunohistochemical observation There was a differential distribution of melatonin receptor types in lung tissue of P. asiatica. Mel1a with immunoreactions of high intensity was found in the bronchial region of the lungs, especially in the apical margins of bronchial mucosal cells and on the finger-like projections of the mucosal foldings, whereas Mel1a with immunoreactions of low intensity was found in other regions of the lung tissue (Figs. 2c, 2d). Some lymphocytes in BALT nodules, as well as in free forms, were also immunopositive. Smooth muscles showed high Mel1a immunoreactions during both reproductive phases. Low intensity of Mel1b immunoreactions was recorded in bronchial mucosal foldings, rather than in other

areas of the lung tissue (Figs. 2e, 2f). When the intensity and occurrence of the immunoreactions for Mel1a were compared with those for Mel1b, we found fewer Mel1a immunoreactive cells in BALT nodules during both reproductive phases. %SR of lymphocytes isolated from lung tissue Isolated lung lymphocytes of P. asiatica during RIP when challenged with Concanavalin A showed significantly high %SR (p < 0.01) compared with isolated lung lymphocytes challenged with Concanavalin A during RAP (Fig. 3b). Hormonal analysis Variations in plasma melatonin and testosterone levels were found during both reproductive phases. Significantly high levels of peripheral melatonin (p < 0.01) were recorded during RIP (200 pg/mL), whereas low levels of peripheral melatonin were recorded during RAP (33.6 pg/mL) (Fig. 4a). Significantly high levels of testosterone (p < 0.01) were recorded during RAP (58.72 pg/mL) than during RIP (10.30 ng/mL) (Fig. 4b). Western blot analysis Densitometric analysis of Western blot with Scion Image software for melatonin receptor types from isolated lung proteins showed more expression of Mel1b than Mel1a during both reproductive phases, whereas the expression of both Published by NRC Research Press

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Fig. 2. Transverse section of lung tissue of male Jungle-Bush Quail (Perdicula asiatica) showing (a) primary bronchus (pbr) with BALT nodule (bn) (hematoxylin and eosin staining) during the reproductively inactive phase (RIP), (b) primary bronchus (pbr) with BALT nodule (bn) (hematoxylin and eosin staining) during the reproductively active phase (RAP), (c) serial section of panel a showing Mel1a receptor immunoreactions counterstained with hematoxylin, (d) serial section of panel b showing Mel1a receptor immunoreactions counterstained with hematoxylin, (e) serial section of panel a showing Mel1b receptor immunoreactions, and (f) serial section of panel b showing Mel1b receptor immunoreactions.

Mel1a and Mel1b receptor types was low during RAP than during RIP (Fig. 5).

Discussion Many avian species do modify their physiological state on a seasonal basis, and are believed to utilize the pineal gland

and its hormone melatonin as a cue to prepare and to respond to the upcoming seasons (Singh and Haldar 2007). Perdicula asiatica is a summer breeder and the activity of their pineal gland is opposite of the activity of their gonads (Haldar and Ghosh 1990). These wild birds suffer more drastic changes in environment compared with domestic poultry such as chickens and turkeys that are confined to a Published by NRC Research Press

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Fig. 3. (a) Morphometric analysis of the variation in size of BALT nodule (bn) and non-BALT nodule (nbn) during the reproductively inactive phase (RIP) and during the reproductively active phase (RAP) of male Jungle-Bush Quail (Perdicula asiatica). **, p < 0.01 (bn vs. bn and nbn vs. nbn during RIP and RAP). a, p < 0.01 (bn vs. nbn during RAP). (b) Percent stimulation ratio (%SR) of lung lymphocytes of male P. asiatica) during RIP and RAP. **, p < 0.01 (%SR during RIP vs. during RAP). Histograms in both panels represent mean ± SE (n = 7 for each group within this experiment).

Fig. 4. Radioimmunoassay showing variations in levels of (a) plasma melatonin and (b) plasma testosterone during the reproductively inactive phase (RIP) and during the reproductively active phase (RAP) of male Jungle-Bush Quail (Perdicula asiatica). Histograms in both panels represent mean ± SE. (a) **, p < 0.01 (plasma melatonin during RIP vs. during RAP). (b) **, p < 0.01 (plasma testosterone during RIP vs. during RAP).

particular condition. Several stressful environmental conditions such as reduced food availability, low ambient temperature, overcrowding, lack of shelter, or increased number of predator do influence seasonal fluctuation in immune function among individuals (Nelson 2004). Avian BALT and non-BALT nodules play a crucial role in the development of local immune responses to inhaled antigens (Bienenstock et al. 1973b; Fagerland and Arp 1990). But the structures of BALT and non-BALT nodules were more frequently studied in chickens and turkeys (Jeurissen et al. 1994; Reese et al. 2006), thus limiting our knowledge to domestic poultry. Localization of BALT and non-BALT nodules and expression of Mel1a and Mel1b were observed in lungs of P. asiatica (Kharwar and Haldar 2010); however, their variation with respect to season has not been well studied. As reported above, we observed a structural similarity in BALT and non-BALT nodules of P. asiatica to that of chickens and turkeys (Fagerland and Arp 1990, 1993; Jeurissen et al. 1994; Reese et al. 2006). The distribution of highly organized BALT nodules and diffusely distributed non-BALT nodules differed a lot, being higher in P. asiatica than in domestic poultry. This might be due to the lungs of a wild bird being more frequently exposed to

environmental threats, thus developing sufficient numbers of BALT nodules. We observed BALT nodules almost in all the junctions of primary bronchus and secondary bronchi, whereas non-BALT nodules were more and diffusely distributed throughout the lungs of P. asiatica. A significant difference in the histology of BALT nodules (i.e., a large number of macrophages) during RAP than during RIP was observed. These induced macrophages could be due to a high level of circulatory gonadal and adrenal steroids (glucocorticoids) during RAP (Sudhakumari et al. 2001; Singh and Haldar 2007) acting as an immunosuppressor. During RAP, while feeding on grains (leftover in the field after harvesting), the pathogens invade the respiratory tract of P. asiatica, which cause an increase in the number of macrophages. A recent study showing this relationship has been documented in our laboratory (R.K. Kharwar and C. Haldar, unpublished data). Furthermore, the high circulatory level of gonadal steroids during RAP reduces the general immune status of P. asiatica (Singh and Haldar 2005) and might have influenced the BALT nodules structurally and functionally. Taking environmental factors into account, i.e., during the summer months of RAP, a long photoperiod has been shown to suppress pineal activity and thus the level of circulating Published by NRC Research Press

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Fig. 5. (a) Western blot showing Mel1a and Mel1b receptor expression in lung tissue of male Jungle-Bush Quail (Perdicula asiatica) during the reproductively inactive phase (RIP) and during the reproductively active phase (RAP). b-actin is used as a loading control. (b) Histogram (mean ± SE) showing comparative percent band intensity of Mel1a and Mel1b receptor types during both RIP and RAP following Scion Image analysis. **, p < 0.01 (Mel1a vs. Mel1b during RIP and RAP). a, p < 0.01 (Mel1a during RIP vs. during RAP). b, p < 0.01 (Mel1b during RIP vs. during RAP).

melatonin, which then increased circulatory levels of testosterone. Testosterone (a steroid hormone) thus suppressed lung-associated immune parameters during RAP. In winter, a short photoperiod elevated pineal activity and thus the circulatory levels of melatonin, which induced lung-associated immunity as shown by the larger sizes of BALT and nonBALT nodules and lymphocyte proliferation (%SR) during RIP. Thus, the correlation between photoperiod length and circulatory levels of melatonin and pineal activity during RAP and RIP is a highly important seasonal adaptation that affects the health of wild seasonal breeders because the enhanced melatonin counteracted the winter-associated stressors (restricted food, low ambient temperature) by inducing immunity (Demas and Nelson 1996). Surprisingly in the Red Knot (Calidris canutus (L., 1758)), Buehler et al. (2009) found no such correlation and suggested that immunocompromise should be correlated with the severity of the environment rather than the time of year. During RAP (summer days, i.e., long photoperiod), higher levels of gonadal steroids in the circulation were responsible for reproductive activity on the one hand and decreased immune status on the other hand (Schuurs and Verheul 1990; Singh and Haldar 2005; Weil et al. 2006). Therefore, the seasonal level of circulating melatonin acts as a major temporal synchron-

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izer that maintains not only the seasonal reproduction but also the immune adaptability of P. asiatica. Our results also indicated that the lymphoid nodules observed in the lungs of P. asiatica during a season could be a major site of induction and amplification of the local immune response of birds, because inhaled antigens can be taken up through its respiratory epithelium and presented to the lymphocytes in BALT nodules for neutralization (Ra´cz et al. 1977; Watanabe et al. 1989). Persistent respiratory inflammation elicited by antigen-specific immunologic reaction, in addition to repeated direct stimulation by a causative antigen, may lead to enlargement of BALT nodules in birds (Cornes 1965; Fagerland and Arp 1993). Studies are required to determine the precise mechanism and causative agents (environmental factors and antigens) involved in development of BALT nodules. We noticed that fluctuation in melatonin and melatonin receptor expression might be responsible for the adaptive evolution in the size and hence number of BALT and non-BALT nodules during unfavorable conditions for the wild avian species. We explored the functional integrity of BALT and nonBALT nodules by performing immunohistochemistry and Western blot analysis for differential expression of melatonin receptor types Mel1a and Mel1b in order to suggest a direct action of melatonin on LAIS. Both receptors Mel1a and Mel1b were present in lung tissue, although their distribution and location were quite different (Figs. 2a–2f). High immunoreactivity for Mel1a on the smooth muscles and mucosal folds were observed. On the other hand, Mel1b immunoreactions was rare on the bronchus region but was more prominent in the interatrial septum of the lung. There were small unidentified cells stained positive for Mel1a, as well as Mel1b, scattered throughout the entire interatrial region. Those cells could be considered phenotypically and morphologically not so identical with the cells observed in LAIS. Western blot analysis of isolated protein from the lungs of P. asiatica during RAP and RIP showed more immunoreactions in term of percent expression of Mel1b compared with Mel1a. Data from the Western blot analysis supported our data from the morphometric analysis of the size of BALT and non-BALT nodules during RIP and RAP. Sufficient distribution of melatonin receptor types in lung tissue suggests that the neuroendocrine system (pineal activity and melatonin) is certainly involved in the maintenance of lung-associated immunity of birds. It could be that melatonin utilizes both kinds of receptors (Mel1a and Mel1b) to regulate the LAIS in P. asiatica. Although our study will contribute to morphological and immunological aspects of LAIS in wild birds, it is imperative that more functional studies be carried out to support some of our conclusions and observations during the different seasons or reproductive phases. On the other hand, use of melatonin and its receptor antagonist during different reproductive phases may further clarify the role of melatonin in the modulation of LAIS, which is our future goal.

Acknowledgements Financial support was provided by the Council of Scientific and Industrial Research (CSIR), New Delhi, and an instrument gift was provided by the Alexander von Humboldt Foundation, Germany. Published by NRC Research Press

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17 Haldar, C., and Ghosh, M. 1990. Annual pineal and testicular cycle in the Indian jungle bush quail, Perdicula asiatica, with reference to the effect of pinealectomy. Gen. Comp. Endocrinol. 77(1): 150–157. doi:10.1016/0016-6480(90)90216-9. Jeurissen, S.H., Vervelde, L., and Janes, E.M. 1994. Structure and function of lymphoid tissues of the chicken. In Poultry science reviews. Vol. 5. Edited by R. Dietert. Elsevier Applied Science, Oxford. pp. 183–207. Kharwar, R.K., and Haldar, C. 2010. Anatomical and histological profile of bronchus-associated lymphoid tissue and localization of melatonin receptor types (Mel1a and Mel1b) in the lung-associated immune system of a tropical bird, Perdicula asiatica. Acta Histochem.. doi:10.1016/j.acthis.2010.01.003. Lee, K.A. 2006. Linking immune defenses and life history at the levels of the individual and the species. Integr. Comp. Biol. 46(6): 1000–1015. doi:10.1093/icb/icl049. Linden, M., and Møller, A.P. 1989. Cost of reproduction and covariation of life history traits in birds. Trends Ecol. Evol. 4(12): 367–371. doi:10.1016/0169-5347(89)90101-8. Martin, L.B., II, Weil, Z.M., and Nelson, R.J. 2006. Refining approaches and diversifying directions in ecoimmunology. Integr. Comp. Biol. 46(6): 1030–1039. doi:10.1093/icb/icl039. Nelson, R.J. 2004. Seasonal immune function and sickness responses. Trends Immunol. 25(4): 187–192. doi:10.1016/j.it. 2004.02.001. PMID:15039045. Pauly, J.L., and Sokal, J.E. 1972. A simplified technique for in vitro studies of lymphocyte reactivity. Proc. Soc. Exp. Biol. Med. 140(1): 40–44. PMID:5033116. Ra´cz, P., Tenner-Ra´cz, K., Myrvik, Q.N., and Fainter, L.K. 1977. Functional architecture of bronchial associated lymphoid tissue and lymphoepithelium in pulmonary cell-mediated reactions in the rabbit. J. Reticuloendothel. Soc. 22(1): 59–83. PMID: 330856. Reese, S., Dalamani, G., and Kaspers, B. 2006. The avian lungassociated immune system: a review. Vet. Res. 37(3): 311–324. doi:10.1051/vetres:2006003. PMID:16611550. Rodriguez, C., Mayo, J.C., Sainz, R.M., Antolı´n, I., Herrera, F., Martı´n, V., and Reiter, R.J. 2004. Regulation of antioxidant enzymes: a significant role for melatonin. J. Pineal Res. 36(1): 1–9. doi:10.1046/j.1600-079X.2003.00092.x. PMID:14675124. Rollag, M.D., and Niswender, G.D. 1976. Radioimmunoassay of serum concentrations of melatonin in sheep exposed to different lighting regimens. Endocrinology, 98(2): 482–488. doi:10.1210/ endo-98-2-482. PMID:1248456. Roy, D., Angelini, N.L., Fujieda, H., Brown, G.M., and Belsham, D.D. 2001. Cyclical regulation of GnRH gene expression in GT1-7 GnRH-secreting neurons by melatonin. Endocrinology, 142(11): 4711–4720. doi:10.1210/en.142.11.4711. PMID: 11606436. Sato, J., Chida, K., Suda, T., Sato, A., and Nakamura, H. 2000. Migratory patterns of thoracic duct lymphocytes into bronchusassociated lymphoid tissue of immunized rats. Lung, 178(5): 295–308. doi:10.1007/s004080000033. PMID:11147313. Savaskan, E., Wirz-Justice, A., Olivieri, G., Pache, M., Kra¨uchi, K., Brydon, L., Jockers, R., Mu¨ller-Spahn, F., and Meyer, P. 2002. Distribution of melatonin MT1 receptor immunoreactivity in human retina. J. Histochem. Cytochem. 50(4): 519–526. PMID:11897804. Schuurs, A.H., and Verheul, H.A.M. 1990. Effects of gender and sex steroids on the immune response. J. Steroid Biochem. 35(2): 157–172. doi:10.1016/0022-4731(90)90270-3. PMID: 2407902. Singh, S.S., and Haldar, C. 2005. Melatonin prevents testosteroneinduced suppression of immune parameters and splenocyte proPublished by NRC Research Press

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