Morphological evidence of immunosenescence in Boa constrictor?

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Eva Dervas, Udo Hetzel, Anja Kipar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6171887/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Jun, 2025 Read the published version in Immunity & Ageing → Version 1 posted 9 You are reading this latest preprint version Abstract Ageing is a complex biological process associated with the decline in immune function, known as immunosenescence, which leads to increased vulnerability to infections and other immune-related diseases. Immunosenescence is a focus of research in mammals and has been particularly well studied in laboratory rodents. However, whether the phenomenon is also a feature in poikilothermic animals such as reptiles, has not been investigated so far. This study explored the lymphoid tissue (spleen and thymus) of Boa constrictor , a boid snake indigenous to South and Central America and Mexico, but widely kept in captivity all over the world, for potential age-related changes. We observed a significant decrease in cellularity in the spleen, coupled with an increase in organ size correlated with age. In both spleen and thymus the connective tissue of capsule and trabeculae increased significantly with age, indicative of progressive fibrosis. In addition, several changes were observed with increasing frequency in older animals, epithelial hyperplasia in the thymic medulla as well stromal fibrosis and an increasing infiltration by so-called granular cells in both organs. Granular cells likely represent a leukocyte subtype; their presence indicates a progressive chronic low-grade inflammatory state in the lymphoid organs, a feature known as inflammageing in other animal classes. They may also play a role in the progressive fibrosis of the connective tissue. The results provide first evidence of immunosenescence in B. constrictor and indicate similarities in the underlying processes across animal classes. Immunosenescence Boa constrictor ageing reptile thymus spleen progressive fibrosis inflammageing Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Ageing is a complex biological process characterized by the progressive decline of physiological functions and increasing cellular damage [ 1 ]. One key aspect of ageing is immunosenescence, a state of dysregulated immune function that occurs with advanced age and has been linked to increased susceptibility to infection, autoimmune diseases, and neoplasia-related mortality in humans [ 2 ]. Immunosenescence has been best studied in mammals (e.g. humans, dogs, mice), however the relationship between ageing and immune function in other vertebrates is far less understood [ 2 , 3 ]. While ageing of reptiles has generally not been a focus of research, however, recently, the interest in comparing the longevity of endotherms (e.g., mammals) and ectotherms (e.g. reptiles) has increased [ 4 – 6 ]. This broadening of interest is driven by the fact that ectotherms hold many animal longevity and reproductive activity records, which highlights their value to improve the understanding of the mechanisms underlying lifespan extension and age-related physiological processes [ 4 , 7 ]. It is important to note that, from an evolutionary perspective, reptiles are the sole surviving ectothermic amniotes and represent a link between ectothermic anamniotic fish and amphibians, and endothermic amniotic birds and mammals [ 8 ]. The reptile immune system has several features in common with its mammalian counterpart; this refers to certain components of both the innate (e.g., complement system, lysozymes, antimicrobial peptides) and adaptive immune response (e.g. T cell receptors, antibody production) that have been described at molecular and/or functional level in various reptile species [ 9 ]. However, research has also highlighted fundamental differences between the immune responses of mammals and reptiles. A notable example is the adaptive immune response in reptiles, which appears to be less specific than that of mammals [ 9 ]. Indeed, reptiles require substantially more time for antibody release and appear to generate very limited immunological memory [ 9 , 10 ]. Moreover, reptiles have neither lymph nodes nor a Bursa Fabricii, although in some species specialized lymphoid structures, such as oesophageal tonsils in boid snakes [ 11 ]and lymphoid axillar structures in geckos [ 12 ], have been described. The reptilian spleen, which would hence be the expected site of B cell proliferation, activation and differentiation, lacks germinal centres; indeed, the most relevant parts of the B cell driven component of the specific immune response is still obscure in reptiles [ 9 , 13 , 14 ]. The boa constrictor ( B. constrictor ), a non-venomous boid snake native to Central and South America, is one of the most popular reptile species in the pet trade, primarily due to its adaptability to environmental conditions and relatively low-maintenance husbandry requirements [ 15 ]. It is also a snake species known for its rather long lifespan, averaging approximately 20 years in the wild and reaching 25 to 35 years in captivity [ 16 , 17 ]. In a recent in-depth study, we described the morphology and composition of the haemolymphatic tissues in B. constrictor [ 14 ]. To characterize the cellular components in situ , we applied immunohistochemistry to detect T cells and macrophages, RNA- in situ hybridization to highlight B cells, and special stains for connective tissue components such as collagen and reticulin. Our findings indicated that the spleen of B. constrictor is predominantly composed of T cells and is devoid of a red pulp [ 14 ]. The thymus was found to persist well beyond the age of sexual maturity; however, it exhibited an age-related decline in total cellularity, resembling the thymic involution observed in many mammals [ 14 ]. Since we observed anecdotal, potential age-related changes in the studied cohort but could not find much information on ageing and age-related features of the immune system of reptiles, we now undertook a more in’depth morphological study on thymus and spleen over the lifespan of boa constrictors, for evidence of immunosenescence. Material and Methods Study animals The study was retrospectively undertaken on 48 B. constrictor from private owners, breeding collections and a reptile shelter. All animals had been submitted for a diagnostic postmortem examination by the owner; the latter was performed upon the owners’ request. Most boas (n = 44) had been submitted for euthanasia, the remaining two (Nos 14, 15) had died naturally and were examined to determine the cause of death. The majority of the snakes originated from collections in which Boid Inclusion Body Disease (BIBD) had previously been diagnosed in one or several animals. The owners submitted the snakes to gain information on the general health status of the collection across age and sex groups (presence of BIBD/reptarenavirus infection or other viral/bacterial diseases of which the owners might not have been aware). Twenty animals (10.1 to 10.20) originated from a reptile shelter, a Switzerland-based volunteer organization that accepts reptiles from private owners or breeders who are unable to maintain their collection. These snakes were euthanized as the shelter had not succeeded in rehoming the animals within a prolonged period (years). For these individuals, information on their exact origin was not available. For the present study only animals without significant pathological changes in the internal organs were chosen, to exclude any underlying diseases that could have affected the composition of the lymphatic tissue. Information on individual animals is provided in Table S1 . Euthanasia, gross and histological examination All snakes submitted alive (n = 46) were euthanized by veterinarians according to the ASPA, (Animals Scientific Procedures Act) 1986, schedule 1 (appropriate methods of humane killing, http://www.legislation.gov.uk/ukpga/1986/14/schedule/1 ) procedure upon the owner’s request, following previously described protocols [ 14 ]. All snakes underwent a full postmortem examination. Samples from all major organs and tissues were collected for histological examination. These were fixed in 10% buffered formalin for appr. 24 h, trimmed and routinely embedded in paraffin wax. Sections (2–3 µm) were prepared and stained with haematoxylin and eosin (HE). Histological examination was performed on all internal organs, relevant diagnoses are compiled in Table S1 . Spleen and/or thymus were examined in more detail, when available (spleen: n = 39; thymus: n = 24). In selected cases, consecutive sections were subjected to special stains to further characterize the components of the connective tissue in capsule and trabeculae: the Van Giesson stain served to highlight collagen fibers, the Reticulin stain for reticulin fibers, and the Resorcinfuchsin stain for elastic fibers, following previously published protocols [ 14 ]. BIBD was excluded in all animals by histological examination of HE stained sections of the internal organs (no evidence of the pathognomonic intracytoplasmic inclusion bodies in a wide variety of parenchymal cells) [ 18 , 19 ]. For the majority of the animals, infection with reptarenaviruses, the causative agents of BIBD, had also been ruled out by multiplex PCR as part of another study [ 14 ]. Morphometric analyses Organ size . The spleen of B. constrictor is an ellipsoid organ [ 14 ]. Therefore, to allow a consistent approach to determine the actual organ dimensions, only spleens that had been bisected and longitudinally embedded were included into the study (n = 41). Both the long and short axes were measured using the Linear Measurement Tool in the NDP.view2 Image viewing software (Hamamatsu Photonics). The thymus was not dissected and embedded in a similar, consistent manner, as the organ was often difficult to fully delineate grossly; hence, to avoid inaccurate values, attempts at organ size measurements were abandoned. For morphometric analyses, HE stained sections of spleen and thymus were scanned using a digital slide scanner (NanoZoomer-XR C12000; Hamamatsu, Hamamatsu City, Japan) and evaluated with the computer program VIS (Visiopharm Integrator System, Version 5.0.4. 1382; Visiopharm, Hoersholm, Denmark). Organ cellularity . For the assessment of the overall cellularity of spleen and thymus, the scanned HE stained sections were assessed as previously described [ 14 ]. Briefly, the total area of lymphoid tissue in both organs (cortex and medulla of the thymus; and all lobules/intertrabecular regions of the spleen) was selected manually as a region of interest (ROI). Afterwards, a decision forest classification method was employed for the classification of cells and the results were presented as the total nuclei count per ROI area (in µm 2 ). Thickness of splenic and thymic capsule and trabeculae . The thickness of the capsule and trabeculae was measured in five representative locations of the spleen and thymus using the Linear Measurement Tool. The mean thickness was subsequently calculated for each, the capsule and the trabeculae. Semiquantitative grading of selected histological features . Frequently occurring histological findings in spleen and thymus were assessed semiquantitatively to examine their potential relationship with age. The following grading system was applied: Epithelial hyperplasia of the thymus: The thymic medulla generally exhibits some epithelial cells that form occasional small tubules. When these tubules were more frequent, epithelial hyperplasia was diagnosed, which was graded as mild (+), moderate (++) or severe (+++), with the latter grade, > 90% of the medulla was affected. Granular cells in the spleen and the thymus: The extent of granular cell infiltration was assessed and graded: not observed (-); predominantly (multifocal) single cells and occasional small groups of 2–3 cells (+); moderate number of cells, predominantly in groups (++); and +++ abundant cells, found disseminated in or in close proximity to the connective tissue framework (+++). Stromal fibrosis of the spleen: The extent of connective tissue deposition in the splenic lobules was assessed, in which (-) corresponded to not observed; + to a mild, ++ to a moderate and +++ to focally extensive to diffuse deposition, the latter spanning across two or more trabecules (+++). Statistical analysis All morphometric parameters and the grading of the histological findings were analysed using a GraphPad Prism software (Version 8.0.2., Boston, USA). The level of significance testing was set with a P value of 0.05. Descriptive statistics were applied, and the data were tested for normality by the D'Agostino-Pearson normality test. Since a former study had revealed significant differences in some blood parameters between boas pre- and post-sexual maturity and reproduction, we allocated the animals to the same two age groups (< 3 years of age; prior to definite sexual maturity and reproduction) and adult (≥ 3 years of age; sexually mature, reproducing animals) to assess the lymphatic tissue-related changes, as already applied in a former study [ 20 ]. Additionally, we also assessed differences between males and females to reveal a potential influence of sex on the examined parameters. Normally distributed data were analysed using two-sample t-test, and non-parametric tests (Wicoxon rank-sum/Mann-Whitney) were used for data that were not normally distributed. The arithmetic mean, including confidence intervals, was determined on normally distributed data and medians were reported where non-parametric tests were applied. A linear regression analysis was used to test if age significantly predicted changes in all morphometric parameters. Results Study population The age of the animals included into the study ranged from 3 days to 21 years. Twenty-five were “subadult” (< 3 years of age) and 24 “adult” (≥ 3 years of age; after sexual maturation). Twenty-one snakes were female, 14 male; for 13 juvenile animals, the sex could not be determined due to lack of differentiation of the gonads. Apart from one animal (No 15) that had shown intermittent dyspnea prior to death, the animals had not presented clinical signs prior to euthanasia or death. Pathological changes were rare and restricted to bilateral suppurative spectaculitis (animal No 12.3), acute vertebral fracture (animal No. 16.1) and mild granulomatous pneumonia (animal No 15.1). These conditions were not considered as likely to have affected the lymphatic tissue composition. Individual animal data are provided in Table S1 . Splenic size increases with age while the cellularity decreases The length of the long and short axis served as a proxy for the size of the spleen. Both were significantly greater in the adult snakes (long axis: subadult snakes arithmetic mean = 2880 µm, CI = 2455–3304 µm, adult snakes arithmetic mean = 7372 µm, CI = 6745–8475 µm, with t = 78.309, df = 33, p < 0.0001, short axis: subadult snakes arithmetic mean = 2184 µm, CI = 1676–2693 µm, adult snakes arithmetic mean = 5904 µm, CI = 4837–7446 µm, with t = 6.238, df = 33, p < 0.0001) (Fig. S1 A and B). The linear regression analysis revealed a significant positive correlation between age and splenic size, i.e. long and short axis (R 2 = 0.525, F = 38.63, p < 0.0001and R 2 = 0.503, F = 35.4, p < 0.0001, respectively), indicating progressive growth of the spleen with age (Fig. 1 A). The detailed results are presented in Table S2A and B. Comparison of the cellularity per µm² in the two age groups showed significantly higher values in the subadult snakes (subadult snakes median = 0.018/µm², CI = 0.018–0.019/µm², adult snakes median = 0.016/ µm², CI = 0.015–0.018, with z = 97, p < 0.001) (Fig. S1 C). The linear regression analysis revealed a significant negative correlation between age and splenic cellularity (R 2 = 0.394, F = 25.31, < 0.0001), indicating that as age increases, splenic cellularity decreases (Table S2B and Fig. S1 B). There was no significant difference in the splenic cellularity between female and male animals. Thymic cellularity is lower in older, sexually mature boas For the thymus, comparison of the cellularity per µm² in the two age groups showed significantly higher values in the subadult snakes (subadult snakes median = 0.02/µm², CI = 0.019–0.027/µm², adult snakes median = 0.018/ µm², CI = 0.016–0.02, with z = 16, p < 0.01) (Table S2A and Fig. 2 A). Although the linear regression analysis did not reveal a significant correlation between age and thymic cellularity, there was a trend for its reduction with age (Table S2B; Fig. 2 B). There was no significant difference in the splenic cellularity between female and male animals. The fibrous framework of spleen and thymus increases with age In the spleen, the thickness of both capsule and trabeculae was significantly higher in adult snakes than in subadult snakes (capsule: subadult snakes arithmetic mean = 44.08 µm, CI = 36.21–51.95 µm, adult snakes arithmetic mean = 177.6 µm CI = 122.1-224.8 µm, with t = 5.881, df = 37, p < 0.0001; trabeculae: subadult snakes median = 51.23 µm, CI = 46.5–65.3 µm, adult snakes median = 230 µm, CI = 167.7-277.3 µm, with z = 16, p < 0.0001) (Fig. S1 D and E). The linear regression analysis revealed a significant positive correlation between age and both capsular and trabecular thickness (R 2 = 0.8, F = 151.5, p < < 0.0001 and R 2 = 0.754, F = 113.5, p < < 0.0001, respectively), indicating their progressive thickening with age (Table S2B; Fig. 1 C-K). With the help of the different special stains it was shown that the observed thickening was mainly due to deposition of collagen fibers (making up approximately 90% of the splenic framework), interspersed with reticulin and elastin fibers. The proportion of the different connective tissue fibers did not appear to change with age (Fig. 1 E-G; I-J). The thymus of adult snakes also exhibited significantly thicker capsules and trabeculae (capsule: subadult snakes arithmetic mean = 12.85 µm, CI = 5.519–21.99 µm, adult snakes arithmetic mean = 72.2 µm, CI = 23.19–106.6 µm, with t = 4.845. df = 22. p < 0.0001 and trabeculae: subadult snakes arithmetic mean = 9.07 µm, CI = 6.483–13.82 µm, adult snakes arithmetic mean = 32.05 µm, CI = 18.76–44.89 µm, with t = 6.792. df = 21. p < 0.0001) (Fig. S2A and B). The linear regression analysis revealed a significant positive correlation between age and the capsular and trabecular thickness (R 2 = 0.381, F = 13.56, p < 0.05 and R 2 = 0.564, F = 27.22, p < 0.0001, respectively), indicating their progressive thickening with age (Table S2B; Fig. 2 C). The composition of the framework was similar to the one of the spleen and did not change with increasing age (Fig. 2 D-I). No significant differences between the two sexes were noted in regard to the splenic or thymic framework. Histopathological changes in spleen and thymus and their potential association with age We have recently described the basic architecture and cell composition of spleen and thymus in B. constrictor in detail [ 14 ]. While this study also reported any histopathological changes, their potential association with age was only marginally discussed. Here, we report several histological features in spleen and thymus that appear to be more frequent and rather consistent in older animals. Table S1 B provides a list of the findings and includes a semiquantitative grading of their extent. Granular cell infiltration . In a previous study, we have identified the so-called granular cells (characterised by abundant yellowish to orange-colored granules in their cytoplasm) in thymus and spleen of boa constrictors where they were particularly evident in animals with BIBD. Although we could not determine the exact origin of these cells, we concluded that they are likely leukocytes, which may be present in higher numbers in association with chronic inflammatory disease processes [ 14 ]. Here, we found predominantly subcapsular aggregates of granular cells in both spleen and thymus (Fig. 3 A and B) of most examined animals, and across all age groups (28/41 in the spleen (68.3%), 12/25 in the thymus (48%); age range of affected animals: 3 days to 21 years). The grade of granular cell infiltration was significantly higher in both organs in adult boas compared to subadult boas, with z = 15,50, p < 0.0001 for the spleen and z = 2, p < 0.05 for the thymus (Figs. S1F and 2C, respectively). The highest grade was observed in the four oldest boas, aged 20 and 21 years. Stromal fibrosis in the spleen . In 12/22 (54.5%) adult boas (10–21 years), the spleen exhibited multifocal to coalescing deposits of connective tissue (collagen fibers and fibrocytes) of mild to severe degree; these extended from capsule or trabeculae into the parenchyma and partially replaced the splenic lobules (Fig. 3 C and D). The highest grade was observed in the four oldest boas, aged 20 and 21 years. It is likely that this feature is linked to the progressive thickening of the capsule and trabeculae that is seen with increasing age. Epithelial hyperplasia in the thymic medulla . We have previously reported epithelial hyperplasia in the thymus in adult snakes, characterized by an increased number of cuboidal to columnar epithelial cells within the thymic medulla [ 11 ]. We also observed epithelial hyperplasia in the thymus in the current cohort, in the adult snakes (8/11, 72.7%; age range: 5 to 20 years); it was not present in the thymus of subadult snakes (n = 14). The extent of epithelial hyperplasia varied overall, with both "younger" adult animals (aged 5 to 6 years) and "older" adult snakes (20 years) exhibiting mild, moderate, and severe degrees. The epithelial cells formed small ductal structures that were occasionally slightly dilated (Fig. 3 E and F). The higher occurrence in adult snakes suggests the feature as a condition potentially linked to sexual maturity and age. Capsular adipocyte accumulation . In two animals (Nos 10.15 and 12.2, aged 11 and 21 years; 5.1%), multiple small aggregates of mature adipocytes (fat tissue) were present between the collagen fibers of the splenic capsule (Fig. 4 A). Similar aggregates were observed in the thymic capsule of two further animals (Nos 3 and 10.6, aged 4 and 6 years, 8.3%). Given the varying ages of the affected snakes, this likely represents an incidental change. Splenic nodules . The spleen of 5 animals (Nos. 5.1, 5.2, 10.4, 10.5, 10.19; aged 3 days to 5 years, 12.8%), exhibited singular, well demarcated nodules of splenic lymphatic tissue protruding from the capsule (Fig. 4 B). The nodules were covered by a thin rim of fibrous tissue. Similar to the intracapsular adipocyte accumulations, due to the broad age range of the animals affected, this finding also was considered an incidental lesion. Thymic cysts . In three snakes (Nos 10.19, 10.6, 10.16, 10.19; aged 3 months, 6 and 10 years, 12.5%), the thymus exhibited large cystic cavities (200 µm to 2 mm in diameter) lined by a monolayered cuboidal to columnal epithelium with frequent apical ciliation and mucus globules in the cytoplasm (Fig. 4 C and D). Their lumen contained amorphous pale eosinophilic homogenous to granular (proteinaceous) material, eosinophilic crystals and/or cell debris. Due to its occurrence across a broad age span, this change is interpreted as an incidental finding. Discussion Immunosenescence refers to the age-related decline in the immune system's ability to respond effectively to infections, vaccinations, and other immune challenges, resulting in increased susceptibility to disease and a reduced capacity to mount robust immune responses [ 21 ]. Studies in humans have shown that, on a cellular level, immunosenescence is linked to changes in immune cell populations, such as a reduction in the number of naive T cells and B cells, reduced T cell receptor repertoire diversity, or accumulation of senescent cells [ 2 , 21 , 22 ]. These alterations can impair the formation of immunological memory and the ability of an organism to respond to new antigens [ 1 , 21 ]. In veterinary medicine, research on immunosenescence has mainly focused on mammals, particularly laboratory rodents as models for humans, while the potential effects of ageing on the immune system in other animal classes, notably Reptilia , have so far not been a focus. The present study represents a first attempt to address this knowledge gap by investigating the lymphoid tissue of the Boa constrictor , a boid snake species frequently kept, bred, and traded in captivity. Our study revealed a significant progressive decrease in lymphoid cellularity in the spleen with age, while the size of the organ, as determined by measuring its long and short axes, increased significantly. These findings are consistent with studies in laboratory mice, in which the spleen's weight increased with advancing age, yet exhibited reduced cellularity, primarily attributed to a decrease in lymphocyte numbers [ 20 ]. Ageing in mice was also associated with further splenic alterations, such as an increase in macrophages, a rearrangement of the microenvironment (in particular an increase in reticular cells), and the loss of well-defined follicular structures [ 20 ]. The assessment of such features and hence a comparative approach is not possible in the B. constrictor as the boa spleen lacks a clear, organized structure. To specify, the spleen is entirely devoid of lymphoid follicles (i.e. no evidence of germinal centres, marginal zone, and mantle zone, as typically observed in mammals [ 21 ]) and a red pulp equivalent, as we described in an earlier study [ 14 ]. Furthermore, while T cells and macrophages can be identified by immunohistochemistry with cross reacting antibodies, there seem to be only few, potentially randomly distributed B cells (as shown by RNA-ISH for CD20 mRNA) and hardly any plasma cells (so far, we only found ultrastructural evidence of their existence in boas) [ 14 ]. Given in particular the lack of reliable immunohistochemical markers for all relevant reptilian leukocyte types, we decided to not pursue the assessment of potential age-associated changes in the composition of the lymphoid tissue any further for the current study. In our study, both lymphoid organs exhibited significant alterations in the connective tissue fibre framework, characterized by a progressive thickening of capsule and trabeculae as the boas aged. Although this phenomenon has not been extensively studied across animal species, it is well documented in human medicine, where older individuals show an overall increase in the thickness of the splenic capsule [ 2 , 23 – 25 ]. Interestingly, these studies have indicated that the apparent thickening is primarily due to an increase in collagen fibres, while other connective tissue components, such as reticulin and elastin fibres, decrease in number [ 25 ]. The progressive reduction in elastin fibres in the splenic capsule has been suggested to limit splenic distention and contribute to its involution with ageing in humans [ 25 ]. The present study found no evidence of any significant differences in the proportion of connective tissue components with the fibrous thickening of capsule and trabeculae in spleen and thymus. These findings suggest progressive extracellular matrix (ECM) synthesis and deposition in boas with age but no ECM remodelling and hence possibly a different underlying mechanism compared to humans. One of the most consistent histological findings associated with advancing age in B. constrictor was the presence and number of so-called granular cells in both thymus and spleen. So far, the origin of this cell type is not known. Granular cells do not express known macrophage markers (such as Iba-1 [ 14 ]), and their granules do not yield consistent results with special stains for, e.g., iron, histamine, glycogen, or mucin ([ 14 ]and unpublished data). Nonetheless, the apparent increasing occurrence of these presumable leukocytes in the lymphoid organs is noteworthy considering that "inflammageing” is a key component of immunosenescence in human ageing. The term refers to a state of chronic, low-grade inflammation that tends to intensify with age and is characterized by an increased infiltration of organs with inflammatory cells [ 21 , 22 ]. Especially macrophages seem to play a key role for induction and maintenance of inflammageing, as their dysfunction during ageing reduces their capacity to remove senescent cells from tissues [ 21 , 22 ]. It is worth mentioning that in humans, inflammageing has also been shown to promote the activation of fibroblasts and other ECM-producing cells, leading to the excessive deposition of ECM proteins and fibrosis in internal organs [ 21 ]. In this regard, the high number of granular cells in a predominantly subcapsular localisation in both thymus and spleen in older boas hints towards an association between the infiltration with granular cells and the observed progressive fibrosis. The present study confirmed an overall significant decrease in thymic cellularity in adult compared to subadult boas; however, linear regression analysis only showed a trend towards a negative correlation between thymic cellularity and age, it did not reach statistical significance. Since we could identify the thymus as a distinct organ also in aged snakes, up to 21 years of age, after many years of sexual maturity, these findings might suggest that the "classical" thymic involution, as observed in mammals and birds [ 7 , 27 – 29 ], does not take place in this snake species. The reason for this phenomenon remains unclear. However, given that the lymphoid organs of B. constrictor are predominantly composed of T cells, as outlined in our previous study [ 14 ], it can be speculated that the thymus plays an essential in T cell development, selection and maturation through a boa’s lifetime. We also observed other histological changes in the lymphatic tissue of older boas, such as epithelial hyperplasia in the thymic medulla. We have already described this feature that is common in association with thymic involution in mammals [ 27 – 29 ] in B. constrictor [ 14 ]. In contrast to epithelial hyperplasia, thymic cysts, which are also frequently linked to thymic involution in mammals, were occasionally observed across all age groups in the current study, providing further evidence that “classical” thymic involution does not occur in this snake species. The unilocular, thin-walled nature of these cysts, along with the presence of ciliated epithelium, suggests a congenital nature; indeed, they might arise from remnants of the brachial arch epithelium [ 30 ]. Conclusions In summary, this study provides first evidence of age-associated changes in the immune system of Boa constrictor , a species that, like reptiles in generals, has so far received very limited attention in the context of immunosenescence. Given the high number of species within the class Reptilia (more than 12000 species are known today [ 31 ]), their high biological and life span variability requires a careful approach towards immunosenescence. It indicates that immunosenescence is an evolutionarily old process that occurs across all animal classes, both homeothermic and poikilothermic, and regardless of the weighing and differentiation of the innate and adapted immune response. Future research with better tools to identify leukocyte subtypes in reptiles should focus on identifying the specific role of the different immune cells and in particular also the so-called granular cells, as these could help to further elucidate the process and mechanisms of inflammageing and ECM deposition in ageing reptiles. Declarations Ethics approval and consent to participate All procedures in this study were approved by the institutional review board (MeF-Ethik-2024-01). The terms of service to which owners agree when submitting an animal for a diagnostic post mortem examination include the permission to make use of material from the examination for both teaching and research. An animal experiment license is not required for conducting examinations on diagnostic material. Consent for publication All authors have approved the paper for publication. Competing interests The authors have no conflicts of interest to declare. Funding This work was conducted without external funding. Author Contribution E.D.: Conceptualization, methodology, investigation, writing (original draft, review & editing). U.H.: Writing (review& editing). A.K.: Conceptualization, writing (review & editing), supervision. Acknowledgement The authors are grateful to the technical staff of the Histology Laboratory, Institute of Veterinary Pathology, University of Zurich for excellent technical support. Data availability The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request. References Keshavarz M, Xie K, Bano D, Ehninger D. Aging – What it is and how to measure it. 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Dev Comp Immunol. 1980;4:703–11. 10.1016/s0145-305x(80)80071-1 . Johnston MR. Perivascular lymphoid tissue associated with the axillary lymph sinus and the lateral vein of Gehyra variegata ( Reptilia:Gekkonidae ). J Morphol. 1973;139:431–7. 10.1002/jmor.1051390405 . Zapata AG, Varas A, Torroba M. Seasonal variations in the immune system of lower vertebrates. Trends Immunol. 1992;13:142–7. 10.1016/0167-5699(92)90112-K . Dervas E, Michalopoulou E, Hepojoki J, Thiele T, Baggio F, Hetzel U, Kipar A. Haemolymphatic tissues of captive boa constrictor ( Boa constrictor ): Morphological features in healthy individuals and with boid inclusion body disease. Dev Comp Immunol. 2025;162:105302. 10.1016/j.dci.2024.105302 . Lindemann L, Boa. constrictor. 2009. https://animaldiversity.org/accounts/Boa_constrictor/ . Accessed 3 Feb 2025. O’Shea M. Boas and pythons of the world. Princeton, New Jersey: Princeton University Press; 2007. Stafford P. Pythons and boas. Neptune City. New Jersey: T.F.H. Publications, Inc. Ltd.; 1986. Stenglein MD, Sanders C, Kistler AL, Ruby JG, Franco JY, Reavill DR, et al. Identification, characterization, and in vitro culture of highly divergent arenaviruses from boa constrictors and annulated tree boas: candidate etiological agents for snake inclusion body disease. mBio. 2012;3:e00180–12. 10.1128/mBio.00180-12 . Hetzel U, Sironen T, Laurinmäki P, Liljeroos L, Patjas A, Henttonen H, et al. Isolation, identification, and characterization of novel arenaviruses, the etiological agents of boid inclusion body disease. J Virol. 2013;87:10918–35. 10.1128/JVI.01123-13 . Dervas E, Liesegang A, Novacco M, Schwarzenberger F, Hetzel U, Michalopoulou E, Kipar A. Haematology, biochemistry and morphological features of peripheral blood cells in captive Boa constrictor : Oxford University Press; 2023. Liu Z, Liang Q, Ren Y, Guo C, Ge X, Wang L, et al. Immunosenescence: molecular mechanisms and diseases. Signal Transduct Target Ther. 2023;8:200. 10.1038/s41392-023-01451-2 . Chinn IK, Blackburn CC, Manley NR, Sempowski GD. Changes in primary lymphoid organs with aging. Semin Immunol. 2012;24:309–20. 10.1016/j.smim.2012.04.005 . Rodrigues CJ, Sacchetti JC, Rodrigues AJ. Age-related changes in the elastic fiber network of the human splenic capsule. Lymphology. 1999;32:64–9. Higginson SM, Sheets NW, Sue LP, Wolfe MM, Kwok AM, Dirks RC, et al. Changes in splenic capsule with aging; beliefs and reality. Am J Surg. 2020;220:178–81. 10.1016/j.amjsurg.2019.09.035 . Al’fonsova EV. Functional morphology of conjunctive tissue stroma of spleen in the age aspect. Adv Gerontol. 2012;25:415–21. Ciriaco E, Píñera PP, Díaz-Esnal B, Laurà R. Age-related changes in the avian primary lymphoid organs (thymus and bursa of Fabricius). Microsc Res Tech. 2003;62:482–7. 10.1002/jemt.10416 . Quaglino D, Capri M, Bergamini G, Euclidi E, Zecca L, Franceschi C, Ronchetti IP. Age-dependent remodeling of rat thymus. Morphological and cytofluorimetric analysis from birth up to one year of age. Eur J Cell Biol. 1998;76(2):156–66. https://doi.org/10.1016/S0171-9335(98)80029-0 . Maxie MG, editor. 2016. Jubb, Kennedy & Palmer’s Pathology of Domestic Animals: Vol 3 (6th edition). W.B. Saunders, pp. 102–268. Haley PJ. The lymphoid system: a review of species differences. J Toxicol Pathol. 2017; 30 (2), 111–123. https://doi.org/0.1293/tox.2016-0075. Maxie MG, editor. 2016. Jubb, Kennedy & Palmer’s Pathology of Domestic Animals: Vol 3 (6th edition). W.B. Saunders, pp. 144–145. Uetz P, Etzold T. The EMBL/EBI Reptile Database. Herpet Rev. 1996;27:174–5. Additional Declarations No competing interests reported. Supplementary Files Supplementalmaterial.docx Cite Share Download PDF Status: Published Journal Publication published 19 Jun, 2025 Read the published version in Immunity & Ageing → Version 1 posted Editorial decision: Revision requested 26 Apr, 2025 Reviews received at journal 23 Apr, 2025 Reviews received at journal 16 Apr, 2025 Reviewers agreed at journal 09 Apr, 2025 Reviewers agreed at journal 24 Mar, 2025 Reviewers invited by journal 13 Mar, 2025 Editor assigned by journal 07 Mar, 2025 Submission checks completed at journal 07 Mar, 2025 First submitted to journal 06 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6171887","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":425468802,"identity":"850bcb0b-6923-4f9d-a315-f6f6543cc5b0","order_by":0,"name":"Eva Dervas","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYLACHjDJfICBsYGBwYAELWwJJGvhMSBOC79E8gOGNxX3Evv7z3yT/LmDQd6ckBbJGWkGjHPOFCfOuJG7TULyDIPhzgYCWgzOHDBg5m1LMGa4wbtNwrCNIcHgAAEt9meOf2Dm/ZdgLH/+zDOJRGK0GLD3AG1pSJAzOJDDJnGQGC0Sx3sKDs45liBneCPN2LKxTcJwAyEt/M3sGx+8qUngkTt/+OHNn2028gRtAQFkNRJEqB8Fo2AUjIJRQBAAAIPRP3xJXA8fAAAAAElFTkSuQmCC","orcid":"","institution":"University of Switzerland","correspondingAuthor":true,"prefix":"","firstName":"Eva","middleName":"","lastName":"Dervas","suffix":""},{"id":425468803,"identity":"52a18310-62b1-4f9e-9052-4f928832a055","order_by":1,"name":"Udo Hetzel","email":"","orcid":"","institution":"University of Switzerland","correspondingAuthor":false,"prefix":"","firstName":"Udo","middleName":"","lastName":"Hetzel","suffix":""},{"id":425468807,"identity":"a5509f6f-0afa-4ebc-ad07-daaa6ba8e33d","order_by":2,"name":"Anja Kipar","email":"","orcid":"","institution":"University of Switzerland","correspondingAuthor":false,"prefix":"","firstName":"Anja","middleName":"","lastName":"Kipar","suffix":""}],"badges":[],"createdAt":"2025-03-06 15:23:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6171887/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6171887/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12979-025-00519-7","type":"published","date":"2025-06-19T15:57:16+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":78121493,"identity":"70248408-f87a-44d7-b4af-f770a5a76561","added_by":"auto","created_at":"2025-03-10 07:10:01","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1529903,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eB. constrictor\u003c/em\u003e, spleen. Morphological and quantitative age-associated changes. \u003cstrong\u003eA. Splenic size.\u003c/strong\u003e The size of the spleen (determined based on the length of both the long (green) and the short (blue) axis in HE stained cross sections) increases with advancing age, as shown by the positive correlation in the linear regression analysis (R² = 0.525, p\u0026lt;0.0001 and R² = 0.503, p\u0026lt;0.0001, respectively). \u003cstrong\u003eB. Splenic cellularity.\u003c/strong\u003e The total number of the cells per lymphoid tissue area decreases with advancing age, as indicated by the negative correlation in the linear regression analysis (R² = 0.396, p\u0026lt;0.0001). \u003cstrong\u003eC. Fibrous framework\u003c/strong\u003e. The thickness of both the capsule and the trabeculae increases with advancing age, as indicated by the positive correlation in the linear regression analysis (R² = 0.799, p\u0026lt;0.0001 and R² = 0.754, p\u0026lt;0.0001, respectively). \u003cstrong\u003eD-H. Morphological features of the spleen\u003c/strong\u003e. \u003cstrong\u003eD-G. Spleen of a subadult boa \u003c/strong\u003e(animal 10.1; 3 months). \u003cstrong\u003eD.\u003c/strong\u003e Cross section, showing densely cellular lymphatic tissue aggregates (asterisk) with thin fibrous capsule and trabeculae. HE stain, bar = 500 µm. \u003cstrong\u003eE-G.\u003c/strong\u003e Closer view of the fibrous framework, with a thin capsule and branching trabeculae (E: HE stain), mainly comprised of collagen fibers (F: Van Giesson stain, red fibers) with fewer embedded elastic fibers (G: Reticulin stain). Bars = 50 µm. \u003cstrong\u003eH-K.\u003c/strong\u003e \u003cstrong\u003eSpleen of an adult boa\u003c/strong\u003e (animal 14.1; 16 years). \u003cstrong\u003eH.\u003c/strong\u003e Cross section, showing that the organ is larger than in the subadult snake and exhibits prominent capsule and trabeculae. HE stain, bar = 500 µm. \u003cstrong\u003eI-K.\u003c/strong\u003e Closer view of the fibrous framework, with a thick capsule and branching trabeculae (I: HE stain), mainly comprised of collagen fibers (J: Van Giesson stain) with fewer embedded elastic fibers (K: Reticulin stain (K). Bars = 50 µm.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6171887/v1/a6c3b6016c75e3d331b887a0.jpeg"},{"id":78121495,"identity":"eef40eb4-300f-4273-ad49-569ae37a9347","added_by":"auto","created_at":"2025-03-10 07:10:01","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":148738,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eB. constrictor\u003c/em\u003e, thymus. Morphological and quantitative age-associated changes\u003cstrong\u003e A, B. Thymic cellularity\u003c/strong\u003e\u003cem\u003e. \u003c/em\u003e\u003cstrong\u003eA.\u003c/strong\u003e\u003cem\u003e \u003c/em\u003eAdult boas show significant fewer cells per lymphatic tissue area (cellularity) compared to subadult boas. Box and whisker plot, **p\u0026lt;0.001. \u003cstrong\u003eB.\u003c/strong\u003eThe total number of cells per lymphatic tissue area shows a trend toward a decrease with advancing age, although no statistically significant correlation is observed in the linear regression analysis (R² = 0.17, p = 0.08). \u003cstrong\u003eC. Fibrous framework\u003c/strong\u003e. The thickness of both the capsule and the trabeculae increases with advancing age, as indicated by the positive correlation in the linear regression analysis (R² = 0.33, p\u0026lt;0.01 and R² = 0.43, p\u0026lt;0.001, respectively). \u003cstrong\u003eD-I. Morphological features of the thymus\u003c/strong\u003e. \u003cstrong\u003eD-F. Thymus of a subadult boa \u003c/strong\u003e(animal 10.18, 3 months). Closer view of the fibrous framework, with a thin capsule (D: HE stain), mainly comprised of collagen fibers (E: Van Giesson stain, red fibers) with fewer embedded elastic fibers (F: Reticulin stain). Bars = 50 µm. \u003cstrong\u003eG-I.\u003c/strong\u003e \u003cstrong\u003eThymus of an adult boa\u003c/strong\u003e (animal 10.7, 20 years). Closer view of the fibrous framework, with a thick capsule and branching trabeculae (G: HE stain), mainly comprised of collagen fibers (H: Van Giesson stain) with fewer embedded elastic fibers (I: Reticulin stain (K). Bars = 50 µm.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6171887/v1/fafc2142415d5db9740bff90.jpeg"},{"id":78121497,"identity":"d95dd608-59d0-4235-88c3-d758976b28be","added_by":"auto","created_at":"2025-03-10 07:10:01","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":177720,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAge-related findings in the spleen and thymus of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. constrictor\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. A, B. \u003c/strong\u003eSpleen, granular cell infiltration (animal 10.7; 20 years, severe degree). \u003cstrong\u003eA.\u003c/strong\u003e Granular cell \u0026nbsp;aggregate in the subcapsular region (arrow). \u003cstrong\u003eB.\u003c/strong\u003e Closer view of granular cells found in groups (arrowhead) or as single cells (arrow). \u003cstrong\u003eC, D\u003c/strong\u003e. Spleen, stromal fibrosis (animal 10.14; 21 years, severe degree). Connective tissue (collagen fibres, arrowhead) replaces the lymphatic tissue (asterisks). \u003cstrong\u003eD.\u003c/strong\u003e Closer view of a collagen fiber deposition (arrowhead). Note also the presence of single granular cells (arrow). \u003cstrong\u003eE, F.\u003c/strong\u003e Thymus, epithelial cell hyperplasia (animal 10.20; 18 years, moderate degree). \u003cstrong\u003eE.\u003c/strong\u003e Epithelial cells form tubular structures in the thymic medulla and cortex (arrow). \u003cstrong\u003eF.\u003c/strong\u003e Closer view of the variably sized tubular structures lined by epithelial cells. The lumen contains homogenous eosinophilic (proteinaceous) material (arrow). HE stains, bars = 50 µm.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6171887/v1/cb98a5cdbcf1432807b1321f.jpeg"},{"id":78121505,"identity":"da71a31d-7fc7-48b7-a972-52a4ce7214bb","added_by":"auto","created_at":"2025-03-10 07:10:01","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":722813,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIncidental findings\u003c/strong\u003e \u003cstrong\u003ein the spleen and thymus of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. constrictor\u003c/strong\u003e\u003c/em\u003e. \u003cstrong\u003eA.\u003c/strong\u003e Thymus, intracapsular adipocytes (animal 3.1; 4 years). Accumulations of mature adipocytes (asterisks) in the thymic capsule. \u003cstrong\u003eB\u003c/strong\u003e. Spleen, nodule formation (animal 10.8; 5 years). A nodular accumulation of lymphatic tissue extends from the capsule. \u003cstrong\u003eC, D.\u003c/strong\u003e Thymus, cyst (animal 10.6; 6 years). \u003cstrong\u003eC.\u003c/strong\u003e Overview, showing a large cavity filled with amorphous pale eosinophilic homogenous to granular material (asterisk), demarcated by a fibrous capsule and lymphatic tissue (arrows). \u003cstrong\u003eD.\u003c/strong\u003e The cyst is lined by a columnar epithelium consisting of goblet cells (arrowhead). Note also the fibrous capsule and infiltrating granular cells (arrow). HE stains, A, B, D: bars = 50 µm, C: bar = 500 µm.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6171887/v1/97cba5b0a9a67e2e9a3aa14d.jpeg"},{"id":85231311,"identity":"9104970d-e91b-40d5-b096-0774c9a080fd","added_by":"auto","created_at":"2025-06-23 16:05:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3429590,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6171887/v1/f8e07743-9427-41d6-b5ce-71a0652fb638.pdf"},{"id":78121496,"identity":"a7e9b687-756a-4dc4-aacd-56590cb394bb","added_by":"auto","created_at":"2025-03-10 07:10:01","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":183970,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalmaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6171887/v1/77f62a1342a59bbd23a0a8bb.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Morphological evidence of immunosenescence in Boa constrictor?","fulltext":[{"header":"Background","content":"\u003cp\u003eAgeing is a complex biological process characterized by the progressive decline of physiological functions and increasing cellular damage [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. One key aspect of ageing is immunosenescence, a state of dysregulated immune function that occurs with advanced age and has been linked to increased susceptibility to infection, autoimmune diseases, and neoplasia-related mortality in humans [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Immunosenescence has been best studied in mammals (e.g. humans, dogs, mice), however the relationship between ageing and immune function in other vertebrates is far less understood [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. While ageing of reptiles has generally not been a focus of research, however, recently, the interest in comparing the longevity of endotherms (e.g., mammals) and ectotherms (e.g. reptiles) has increased [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This broadening of interest is driven by the fact that ectotherms hold many animal longevity and reproductive activity records, which highlights their value to improve the understanding of the mechanisms underlying lifespan extension and age-related physiological processes [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. It is important to note that, from an evolutionary perspective, reptiles are the sole surviving ectothermic amniotes and represent a link between ectothermic anamniotic fish and amphibians, and endothermic amniotic birds and mammals [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe reptile immune system has several features in common with its mammalian counterpart; this refers to certain components of both the innate (e.g., complement system, lysozymes, antimicrobial peptides) and adaptive immune response (e.g. T cell receptors, antibody production) that have been described at molecular and/or functional level in various reptile species [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, research has also highlighted fundamental differences between the immune responses of mammals and reptiles. A notable example is the adaptive immune response in reptiles, which appears to be less specific than that of mammals [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Indeed, reptiles require substantially more time for antibody release and appear to generate very limited immunological memory [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Moreover, reptiles have neither lymph nodes nor a Bursa Fabricii, although in some species specialized lymphoid structures, such as oesophageal tonsils in boid snakes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]and lymphoid axillar structures in geckos [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], have been described. The reptilian spleen, which would hence be the expected site of B cell proliferation, activation and differentiation, lacks germinal centres; indeed, the most relevant parts of the B cell driven component of the specific immune response is still obscure in reptiles [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe boa constrictor (\u003cem\u003eB. constrictor\u003c/em\u003e), a non-venomous boid snake native to Central and South America, is one of the most popular reptile species in the pet trade, primarily due to its adaptability to environmental conditions and relatively low-maintenance husbandry requirements [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. It is also a snake species known for its rather long lifespan, averaging approximately 20 years in the wild and reaching 25 to 35 years in captivity [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn a recent in-depth study, we described the morphology and composition of the haemolymphatic tissues in \u003cem\u003eB. constrictor\u003c/em\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. To characterize the cellular components \u003cem\u003ein situ\u003c/em\u003e, we applied immunohistochemistry to detect T cells and macrophages, RNA-\u003cem\u003ein situ\u003c/em\u003e hybridization to highlight B cells, and special stains for connective tissue components such as collagen and reticulin. Our findings indicated that the spleen of \u003cem\u003eB. constrictor\u003c/em\u003e is predominantly composed of T cells and is devoid of a red pulp [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The thymus was found to persist well beyond the age of sexual maturity; however, it exhibited an age-related decline in total cellularity, resembling the thymic involution observed in many mammals [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Since we observed anecdotal, potential age-related changes in the studied cohort but could not find much information on ageing and age-related features of the immune system of reptiles, we now undertook a more in\u0026rsquo;depth morphological study on thymus and spleen over the lifespan of boa constrictors, for evidence of immunosenescence.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy animals\u003c/h2\u003e \u003cp\u003eThe study was retrospectively undertaken on 48 \u003cem\u003eB. constrictor\u003c/em\u003e from private owners, breeding collections and a reptile shelter. All animals had been submitted for a diagnostic postmortem examination by the owner; the latter was performed upon the owners\u0026rsquo; request. Most boas (n\u0026thinsp;=\u0026thinsp;44) had been submitted for euthanasia, the remaining two (Nos 14, 15) had died naturally and were examined to determine the cause of death.\u003c/p\u003e \u003cp\u003eThe majority of the snakes originated from collections in which Boid Inclusion Body Disease (BIBD) had previously been diagnosed in one or several animals. The owners submitted the snakes to gain information on the general health status of the collection across age and sex groups (presence of BIBD/reptarenavirus infection or other viral/bacterial diseases of which the owners might not have been aware). Twenty animals (10.1 to 10.20) originated from a reptile shelter, a Switzerland-based volunteer organization that accepts reptiles from private owners or breeders who are unable to maintain their collection. These snakes were euthanized as the shelter had not succeeded in rehoming the animals within a prolonged period (years). For these individuals, information on their exact origin was not available.\u003c/p\u003e \u003cp\u003eFor the present study only animals without significant pathological changes in the internal organs were chosen, to exclude any underlying diseases that could have affected the composition of the lymphatic tissue. Information on individual animals is provided in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEuthanasia, gross and histological examination\u003c/h3\u003e\n\u003cp\u003eAll snakes submitted alive (n\u0026thinsp;=\u0026thinsp;46) were euthanized by veterinarians according to the ASPA, (Animals Scientific Procedures Act) 1986, schedule 1 (appropriate methods of humane killing, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.legislation.gov.uk/ukpga/1986/14/schedule/1\u003c/span\u003e\u003cspan address=\"http://www.legislation.gov.uk/ukpga/1986/14/schedule/1\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e procedure upon the owner\u0026rsquo;s request, following previously described protocols [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAll snakes underwent a full postmortem examination. Samples from all major organs and tissues were collected for histological examination. These were fixed in 10% buffered formalin for appr. 24 h, trimmed and routinely embedded in paraffin wax. Sections (2\u0026ndash;3 \u0026micro;m) were prepared and stained with haematoxylin and eosin (HE). Histological examination was performed on all internal organs, relevant diagnoses are compiled in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Spleen and/or thymus were examined in more detail, when available (spleen: n\u0026thinsp;=\u0026thinsp;39; thymus: n\u0026thinsp;=\u0026thinsp;24). In selected cases, consecutive sections were subjected to special stains to further characterize the components of the connective tissue in capsule and trabeculae: the Van Giesson stain served to highlight collagen fibers, the Reticulin stain for reticulin fibers, and the Resorcinfuchsin stain for elastic fibers, following previously published protocols [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBIBD was excluded in all animals by histological examination of HE stained sections of the internal organs (no evidence of the pathognomonic intracytoplasmic inclusion bodies in a wide variety of parenchymal cells) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. For the majority of the animals, infection with reptarenaviruses, the causative agents of BIBD, had also been ruled out by multiplex PCR as part of another study [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eMorphometric analyses\u003c/h3\u003e\n\u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eOrgan size\u003c/span\u003e. The spleen of \u003cem\u003eB. constrictor\u003c/em\u003e is an ellipsoid organ [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, to allow a consistent approach to determine the actual organ dimensions, only spleens that had been bisected and longitudinally embedded were included into the study (n\u0026thinsp;=\u0026thinsp;41). Both the long and short axes were measured using the Linear Measurement Tool in the NDP.view2 Image viewing software (Hamamatsu Photonics). The thymus was not dissected and embedded in a similar, consistent manner, as the organ was often difficult to fully delineate grossly; hence, to avoid inaccurate values, attempts at organ size measurements were abandoned.\u003c/p\u003e \u003cp\u003eFor morphometric analyses, HE stained sections of spleen and thymus were scanned using a digital slide scanner (NanoZoomer-XR C12000; Hamamatsu, Hamamatsu City, Japan) and evaluated with the computer program VIS (Visiopharm Integrator System, Version 5.0.4. 1382; Visiopharm, Hoersholm, Denmark).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eOrgan cellularity\u003c/span\u003e. For the assessment of the overall cellularity of spleen and thymus, the scanned HE stained sections were assessed as previously described [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Briefly, the total area of lymphoid tissue in both organs (cortex and medulla of the thymus; and all lobules/intertrabecular regions of the spleen) was selected manually as a region of interest (ROI). Afterwards, a decision forest classification method was employed for the classification of cells and the results were presented as the total nuclei count per ROI area (in \u0026micro;m\u003csup\u003e2\u003c/sup\u003e).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eThickness of splenic and thymic capsule and trabeculae\u003c/span\u003e. The thickness of the capsule and trabeculae was measured in five representative locations of the spleen and thymus using the Linear Measurement Tool. The mean thickness was subsequently calculated for each, the capsule and the trabeculae.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eSemiquantitative grading of selected histological features\u003c/span\u003e. Frequently occurring histological findings in spleen and thymus were assessed semiquantitatively to examine their potential relationship with age. The following grading system was applied:\u003c/p\u003e \u003cp\u003eEpithelial hyperplasia of the thymus: The thymic medulla generally exhibits some epithelial cells that form occasional small tubules. When these tubules were more frequent, epithelial hyperplasia was diagnosed, which was graded as mild (+), moderate (++) or severe (+++), with the latter grade, \u0026gt; 90% of the medulla was affected.\u003c/p\u003e \u003cp\u003eGranular cells in the spleen and the thymus: The extent of granular cell infiltration was assessed and graded: not observed (-); predominantly (multifocal) single cells and occasional small groups of 2\u0026ndash;3 cells (+); moderate number of cells, predominantly in groups (++); and +++ abundant cells, found disseminated in or in close proximity to the connective tissue framework (+++).\u003c/p\u003e \u003cp\u003eStromal fibrosis of the spleen: The extent of connective tissue deposition in the splenic lobules was assessed, in which (-) corresponded to not observed; + to a mild, ++ to a moderate and +++ to focally extensive to diffuse deposition, the latter spanning across two or more trabecules (+++).\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll morphometric parameters and the grading of the histological findings were analysed using a GraphPad Prism software (Version 8.0.2., Boston, USA). The level of significance testing was set with a P value of 0.05. Descriptive statistics were applied, and the data were tested for normality by the D'Agostino-Pearson normality test. Since a former study had revealed significant differences in some blood parameters between boas pre- and post-sexual maturity and reproduction, we allocated the animals to the same two age groups (\u0026lt;\u0026thinsp;3 years of age; prior to definite sexual maturity and reproduction) and adult (\u0026ge;\u0026thinsp;3 years of age; sexually mature, reproducing animals) to assess the lymphatic tissue-related changes, as already applied in a former study [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Additionally, we also assessed differences between males and females to reveal a potential influence of sex on the examined parameters. Normally distributed data were analysed using two-sample t-test, and non-parametric tests (Wicoxon rank-sum/Mann-Whitney) were used for data that were not normally distributed. The arithmetic mean, including confidence intervals, was determined on normally distributed data and medians were reported where non-parametric tests were applied. A linear regression analysis was used to test if age significantly predicted changes in all morphometric parameters.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStudy population\u003c/h2\u003e \u003cp\u003eThe age of the animals included into the study ranged from 3 days to 21 years. Twenty-five were \u0026ldquo;subadult\u0026rdquo; (\u0026lt;\u0026thinsp;3 years of age) and 24 \u0026ldquo;adult\u0026rdquo; (\u0026ge;\u0026thinsp;3 years of age; after sexual maturation). Twenty-one snakes were female, 14 male; for 13 juvenile animals, the sex could not be determined due to lack of differentiation of the gonads. Apart from one animal (No 15) that had shown intermittent dyspnea prior to death, the animals had not presented clinical signs prior to euthanasia or death. Pathological changes were rare and restricted to bilateral suppurative spectaculitis (animal No 12.3), acute vertebral fracture (animal No. 16.1) and mild granulomatous pneumonia (animal No 15.1). These conditions were not considered as likely to have affected the lymphatic tissue composition. Individual animal data are provided in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSplenic size increases with age while the cellularity decreases\u003c/h3\u003e\n\u003cp\u003eThe length of the long and short axis served as a proxy for the size of the spleen. Both were significantly greater in the adult snakes (long axis: subadult snakes arithmetic mean\u0026thinsp;=\u0026thinsp;2880 \u0026micro;m, CI\u0026thinsp;=\u0026thinsp;2455\u0026ndash;3304 \u0026micro;m, adult snakes arithmetic mean\u0026thinsp;=\u0026thinsp;7372 \u0026micro;m, CI\u0026thinsp;=\u0026thinsp;6745\u0026ndash;8475 \u0026micro;m, with t\u0026thinsp;=\u0026thinsp;78.309, df\u0026thinsp;=\u0026thinsp;33, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, short axis: subadult snakes arithmetic mean\u0026thinsp;=\u0026thinsp;2184 \u0026micro;m, CI\u0026thinsp;=\u0026thinsp;1676\u0026ndash;2693 \u0026micro;m, adult snakes arithmetic mean\u0026thinsp;=\u0026thinsp;5904 \u0026micro;m, CI\u0026thinsp;=\u0026thinsp;4837\u0026ndash;7446 \u0026micro;m, with t\u0026thinsp;=\u0026thinsp;6.238, df\u0026thinsp;=\u0026thinsp;33, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA and B). The linear regression analysis revealed a significant positive correlation between age and splenic size, i.e. long and short axis (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.525, F\u0026thinsp;=\u0026thinsp;38.63, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001and R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.503, F\u0026thinsp;=\u0026thinsp;35.4, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, respectively), indicating progressive growth of the spleen with age (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The detailed results are presented in Table S2A and B.\u003c/p\u003e \u003cp\u003eComparison of the cellularity per \u0026micro;m\u0026sup2; in the two age groups showed significantly higher values in the subadult snakes (subadult snakes median\u0026thinsp;=\u0026thinsp;0.018/\u0026micro;m\u0026sup2;, CI\u0026thinsp;=\u0026thinsp;0.018\u0026ndash;0.019/\u0026micro;m\u0026sup2;, adult snakes median\u0026thinsp;=\u0026thinsp;0.016/ \u0026micro;m\u0026sup2;, CI\u0026thinsp;=\u0026thinsp;0.015\u0026ndash;0.018, with z\u0026thinsp;=\u0026thinsp;97, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). The linear regression analysis revealed a significant negative correlation between age and splenic cellularity (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.394, F\u0026thinsp;=\u0026thinsp;25.31, \u0026lt;\u0026thinsp;0.0001), indicating that as age increases, splenic cellularity decreases (Table S2B and Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). There was no significant difference in the splenic cellularity between female and male animals.\u003c/p\u003e\n\u003ch3\u003eThymic cellularity is lower in older, sexually mature boas\u003c/h3\u003e\n\u003cp\u003eFor the thymus, comparison of the cellularity per \u0026micro;m\u0026sup2; in the two age groups showed significantly higher values in the subadult snakes (subadult snakes median\u0026thinsp;=\u0026thinsp;0.02/\u0026micro;m\u0026sup2;, CI\u0026thinsp;=\u0026thinsp;0.019\u0026ndash;0.027/\u0026micro;m\u0026sup2;, adult snakes median\u0026thinsp;=\u0026thinsp;0.018/ \u0026micro;m\u0026sup2;, CI\u0026thinsp;=\u0026thinsp;0.016\u0026ndash;0.02, with z\u0026thinsp;=\u0026thinsp;16, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Table S2A and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Although the linear regression analysis did not reveal a significant correlation between age and thymic cellularity, there was a trend for its reduction with age (Table S2B; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). There was no significant difference in the splenic cellularity between female and male animals.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eThe fibrous framework of spleen and thymus increases with age\u003c/h2\u003e \u003cp\u003eIn the spleen, the thickness of both capsule and trabeculae was significantly higher in adult snakes than in subadult snakes (capsule: subadult snakes arithmetic mean\u0026thinsp;=\u0026thinsp;44.08 \u0026micro;m, CI\u0026thinsp;=\u0026thinsp;36.21\u0026ndash;51.95 \u0026micro;m, adult snakes arithmetic mean\u0026thinsp;=\u0026thinsp;177.6 \u0026micro;m CI\u0026thinsp;=\u0026thinsp;122.1-224.8 \u0026micro;m, with t\u0026thinsp;=\u0026thinsp;5.881, df\u0026thinsp;=\u0026thinsp;37, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; trabeculae: subadult snakes median\u0026thinsp;=\u0026thinsp;51.23 \u0026micro;m, CI\u0026thinsp;=\u0026thinsp;46.5\u0026ndash;65.3 \u0026micro;m, adult snakes median\u0026thinsp;=\u0026thinsp;230 \u0026micro;m, CI\u0026thinsp;=\u0026thinsp;167.7-277.3 \u0026micro;m, with z\u0026thinsp;=\u0026thinsp;16, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD and E). The linear regression analysis revealed a significant positive correlation between age and both capsular and trabecular thickness (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.8, F\u0026thinsp;=\u0026thinsp;151.5, p\u0026thinsp;\u0026lt;\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.754, F\u0026thinsp;=\u0026thinsp;113.5, p\u0026thinsp;\u0026lt;\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, respectively), indicating their progressive thickening with age (Table S2B; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-K). With the help of the different special stains it was shown that the observed thickening was mainly due to deposition of collagen fibers (making up approximately 90% of the splenic framework), interspersed with reticulin and elastin fibers. The proportion of the different connective tissue fibers did not appear to change with age (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-G; I-J).\u003c/p\u003e \u003cp\u003eThe thymus of adult snakes also exhibited significantly thicker capsules and trabeculae (capsule: subadult snakes arithmetic mean\u0026thinsp;=\u0026thinsp;12.85 \u0026micro;m, CI\u0026thinsp;=\u0026thinsp;5.519\u0026ndash;21.99 \u0026micro;m, adult snakes arithmetic mean\u0026thinsp;=\u0026thinsp;72.2 \u0026micro;m, CI\u0026thinsp;=\u0026thinsp;23.19\u0026ndash;106.6 \u0026micro;m, with t\u0026thinsp;=\u0026thinsp;4.845. df\u0026thinsp;=\u0026thinsp;22. p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and trabeculae: subadult snakes arithmetic mean\u0026thinsp;=\u0026thinsp;9.07 \u0026micro;m, CI\u0026thinsp;=\u0026thinsp;6.483\u0026ndash;13.82 \u0026micro;m, adult snakes arithmetic mean\u0026thinsp;=\u0026thinsp;32.05 \u0026micro;m, CI\u0026thinsp;=\u0026thinsp;18.76\u0026ndash;44.89 \u0026micro;m, with t\u0026thinsp;=\u0026thinsp;6.792. df\u0026thinsp;=\u0026thinsp;21. p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig. S2A and B). The linear regression analysis revealed a significant positive correlation between age and the capsular and trabecular thickness (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.381, F\u0026thinsp;=\u0026thinsp;13.56, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.564, F\u0026thinsp;=\u0026thinsp;27.22, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, respectively), indicating their progressive thickening with age (Table S2B; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The composition of the framework was similar to the one of the spleen and did not change with increasing age (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-I). No significant differences between the two sexes were noted in regard to the splenic or thymic framework.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological changes in spleen and thymus and their potential association with age\u003c/h2\u003e \u003cp\u003eWe have recently described the basic architecture and cell composition of spleen and thymus in \u003cem\u003eB. constrictor\u003c/em\u003e in detail [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. While this study also reported any histopathological changes, their potential association with age was only marginally discussed. Here, we report several histological features in spleen and thymus that appear to be more frequent and rather consistent in older animals. Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB provides a list of the findings and includes a semiquantitative grading of their extent.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGranular cell infiltration\u003c/span\u003e. In a previous study, we have identified the so-called granular cells (characterised by abundant yellowish to orange-colored granules in their cytoplasm) in thymus and spleen of boa constrictors where they were particularly evident in animals with BIBD. Although we could not determine the exact origin of these cells, we concluded that they are likely leukocytes, which may be present in higher numbers in association with chronic inflammatory disease processes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Here, we found predominantly subcapsular aggregates of granular cells in both spleen and thymus (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B) of most examined animals, and across all age groups (28/41 in the spleen (68.3%), 12/25 in the thymus (48%); age range of affected animals: 3 days to 21 years). The grade of granular cell infiltration was significantly higher in both organs in adult boas compared to subadult boas, with z\u0026thinsp;=\u0026thinsp;15,50, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 for the spleen and z\u0026thinsp;=\u0026thinsp;2, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for the thymus (Figs. S1F and 2C, respectively). The highest grade was observed in the four oldest boas, aged 20 and 21 years.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eStromal fibrosis in the spleen\u003c/span\u003e. In 12/22 (54.5%) adult boas (10\u0026ndash;21 years), the spleen exhibited multifocal to coalescing deposits of connective tissue (collagen fibers and fibrocytes) of mild to severe degree; these extended from capsule or trabeculae into the parenchyma and partially replaced the splenic lobules (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and D). The highest grade was observed in the four oldest boas, aged 20 and 21 years. It is likely that this feature is linked to the progressive thickening of the capsule and trabeculae that is seen with increasing age.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eEpithelial hyperplasia in the thymic medulla\u003c/span\u003e. We have previously reported epithelial hyperplasia in the thymus in adult snakes, characterized by an increased number of cuboidal to columnar epithelial cells within the thymic medulla [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. We also observed epithelial hyperplasia in the thymus in the current cohort, in the adult snakes (8/11, 72.7%; age range: 5 to 20 years); it was not present in the thymus of subadult snakes (n\u0026thinsp;=\u0026thinsp;14). The extent of epithelial hyperplasia varied overall, with both \"younger\" adult animals (aged 5 to 6 years) and \"older\" adult snakes (20 years) exhibiting mild, moderate, and severe degrees. The epithelial cells formed small ductal structures that were occasionally slightly dilated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and F). The higher occurrence in adult snakes suggests the feature as a condition potentially linked to sexual maturity and age.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCapsular adipocyte accumulation\u003c/span\u003e. In two animals (Nos 10.15 and 12.2, aged 11 and 21 years; 5.1%), multiple small aggregates of mature adipocytes (fat tissue) were present between the collagen fibers of the splenic capsule (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Similar aggregates were observed in the thymic capsule of two further animals (Nos 3 and 10.6, aged 4 and 6 years, 8.3%). Given the varying ages of the affected snakes, this likely represents an incidental change.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eSplenic nodules\u003c/span\u003e. The spleen of 5 animals (Nos. 5.1, 5.2, 10.4, 10.5, 10.19; aged 3 days to 5 years, 12.8%), exhibited singular, well demarcated nodules of splenic lymphatic tissue protruding from the capsule (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The nodules were covered by a thin rim of fibrous tissue. Similar to the intracapsular adipocyte accumulations, due to the broad age range of the animals affected, this finding also was considered an incidental lesion.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eThymic cysts\u003c/span\u003e. In three snakes (Nos 10.19, 10.6, 10.16, 10.19; aged 3 months, 6 and 10 years, 12.5%), the thymus exhibited large cystic cavities (200 \u0026micro;m to 2 mm in diameter) lined by a monolayered cuboidal to columnal epithelium with frequent apical ciliation and mucus globules in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and D). Their lumen contained amorphous pale eosinophilic homogenous to granular (proteinaceous) material, eosinophilic crystals and/or cell debris. Due to its occurrence across a broad age span, this change is interpreted as an incidental finding.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eImmunosenescence refers to the age-related decline in the immune system's ability to respond effectively to infections, vaccinations, and other immune challenges, resulting in increased susceptibility to disease and a reduced capacity to mount robust immune responses [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Studies in humans have shown that, on a cellular level, immunosenescence is linked to changes in immune cell populations, such as a reduction in the number of naive T cells and B cells, reduced T cell receptor repertoire diversity, or accumulation of senescent cells [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These alterations can impair the formation of immunological memory and the ability of an organism to respond to new antigens [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In veterinary medicine, research on immunosenescence has mainly focused on mammals, particularly laboratory rodents as models for humans, while the potential effects of ageing on the immune system in other animal classes, notably \u003cem\u003eReptilia\u003c/em\u003e, have so far not been a focus. The present study represents a first attempt to address this knowledge gap by investigating the lymphoid tissue of the \u003cem\u003eBoa constrictor\u003c/em\u003e, a boid snake species frequently kept, bred, and traded in captivity.\u003c/p\u003e \u003cp\u003eOur study revealed a significant progressive decrease in lymphoid cellularity in the spleen with age, while the size of the organ, as determined by measuring its long and short axes, increased significantly. These findings are consistent with studies in laboratory mice, in which the spleen's weight increased with advancing age, yet exhibited reduced cellularity, primarily attributed to a decrease in lymphocyte numbers [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Ageing in mice was also associated with further splenic alterations, such as an increase in macrophages, a rearrangement of the microenvironment (in particular an increase in reticular cells), and the loss of well-defined follicular structures [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The assessment of such features and hence a comparative approach is not possible in the \u003cem\u003eB. constrictor\u003c/em\u003e as the boa spleen lacks a clear, organized structure. To specify, the spleen is entirely devoid of lymphoid follicles (i.e. no evidence of germinal centres, marginal zone, and mantle zone, as typically observed in mammals [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]) and a red pulp equivalent, as we described in an earlier study [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Furthermore, while T cells and macrophages can be identified by immunohistochemistry with cross reacting antibodies, there seem to be only few, potentially randomly distributed B cells (as shown by RNA-ISH for CD20 mRNA) and hardly any plasma cells (so far, we only found ultrastructural evidence of their existence in boas) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Given in particular the lack of reliable immunohistochemical markers for all relevant reptilian leukocyte types, we decided to not pursue the assessment of potential age-associated changes in the composition of the lymphoid tissue any further for the current study.\u003c/p\u003e \u003cp\u003eIn our study, both lymphoid organs exhibited significant alterations in the connective tissue fibre framework, characterized by a progressive thickening of capsule and trabeculae as the boas aged. Although this phenomenon has not been extensively studied across animal species, it is well documented in human medicine, where older individuals show an overall increase in the thickness of the splenic capsule [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Interestingly, these studies have indicated that the apparent thickening is primarily due to an increase in collagen fibres, while other connective tissue components, such as reticulin and elastin fibres, decrease in number [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The progressive reduction in elastin fibres in the splenic capsule has been suggested to limit splenic distention and contribute to its involution with ageing in humans [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The present study found no evidence of any significant differences in the proportion of connective tissue components with the fibrous thickening of capsule and trabeculae in spleen and thymus. These findings suggest progressive extracellular matrix (ECM) synthesis and deposition in boas with age but no ECM remodelling and hence possibly a different underlying mechanism compared to humans.\u003c/p\u003e \u003cp\u003eOne of the most consistent histological findings associated with advancing age in \u003cem\u003eB. constrictor\u003c/em\u003e was the presence and number of so-called granular cells in both thymus and spleen. So far, the origin of this cell type is not known. Granular cells do not express known macrophage markers (such as Iba-1 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]), and their granules do not yield consistent results with special stains for, e.g., iron, histamine, glycogen, or mucin ([\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]and unpublished data). Nonetheless, the apparent increasing occurrence of these presumable leukocytes in the lymphoid organs is noteworthy considering that \"inflammageing\u0026rdquo; is a key component of immunosenescence in human ageing. The term refers to a state of chronic, low-grade inflammation that tends to intensify with age and is characterized by an increased infiltration of organs with inflammatory cells [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Especially macrophages seem to play a key role for induction and maintenance of inflammageing, as their dysfunction during ageing reduces their capacity to remove senescent cells from tissues [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. It is worth mentioning that in humans, inflammageing has also been shown to promote the activation of fibroblasts and other ECM-producing cells, leading to the excessive deposition of ECM proteins and fibrosis in internal organs [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In this regard, the high number of granular cells in a predominantly subcapsular localisation in both thymus and spleen in older boas hints towards an association between the infiltration with granular cells and the observed progressive fibrosis.\u003c/p\u003e \u003cp\u003eThe present study confirmed an overall significant decrease in thymic cellularity in adult compared to subadult boas; however, linear regression analysis only showed a trend towards a negative correlation between thymic cellularity and age, it did not reach statistical significance. Since we could identify the thymus as a distinct organ also in aged snakes, up to 21 years of age, after many years of sexual maturity, these findings might suggest that the \"classical\" thymic involution, as observed in mammals and birds [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], does not take place in this snake species. The reason for this phenomenon remains unclear. However, given that the lymphoid organs of \u003cem\u003eB. constrictor\u003c/em\u003e are predominantly composed of T cells, as outlined in our previous study [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], it can be speculated that the thymus plays an essential in T cell development, selection and maturation through a boa\u0026rsquo;s lifetime.\u003c/p\u003e \u003cp\u003eWe also observed other histological changes in the lymphatic tissue of older boas, such as epithelial hyperplasia in the thymic medulla. We have already described this feature that is common in association with thymic involution in mammals [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] in \u003cem\u003eB. constrictor\u003c/em\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In contrast to epithelial hyperplasia, thymic cysts, which are also frequently linked to thymic involution in mammals, were occasionally observed across all age groups in the current study, providing further evidence that \u0026ldquo;classical\u0026rdquo; thymic involution does not occur in this snake species. The unilocular, thin-walled nature of these cysts, along with the presence of ciliated epithelium, suggests a congenital nature; indeed, they might arise from remnants of the brachial arch epithelium [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, this study provides first evidence of age-associated changes in the immune system of \u003cem\u003eBoa constrictor\u003c/em\u003e, a species that, like reptiles in generals, has so far received very limited attention in the context of immunosenescence. Given the high number of species within the class Reptilia (more than 12000 species are known today [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]), their high biological and life span variability requires a careful approach towards immunosenescence. It indicates that immunosenescence is an evolutionarily old process that occurs across all animal classes, both homeothermic and poikilothermic, and regardless of the weighing and differentiation of the innate and adapted immune response. Future research with better tools to identify leukocyte subtypes in reptiles should focus on identifying the specific role of the different immune cells and in particular also the so-called granular cells, as these could help to further elucidate the process and mechanisms of inflammageing and ECM deposition in ageing reptiles.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003e All procedures in this study were approved by the institutional review board (MeF-Ethik-2024-01). The terms of service to which owners agree when submitting an animal for a diagnostic post mortem examination include the permission to make use of material from the examination for both teaching and research. An animal experiment license is not required for conducting examinations on diagnostic material.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003e All authors have approved the paper for publication.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors have no conflicts of interest to declare.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was conducted without external funding.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eE.D.: Conceptualization, methodology, investigation, writing (original draft, review \u0026amp; editing). U.H.: Writing (review\u0026amp; editing). A.K.: Conceptualization, writing (review \u0026amp; editing), supervision.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors are grateful to the technical staff of the Histology Laboratory, Institute of Veterinary Pathology, University of Zurich for excellent technical support.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKeshavarz M, Xie K, Bano D, Ehninger D. Aging \u0026ndash; What it is and how to measure it. 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Herpet Rev. 1996;27:174\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"immunity-and-ageing","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"iage","sideBox":"Learn more about [Immunity \u0026 Ageing](http://immunityageing.biomedcentral.com/)","snPcode":"12979","submissionUrl":"https://submission.nature.com/new-submission/12979/3","title":"Immunity \u0026 Ageing","twitterHandle":"@ImmunAllergyBMC","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Immunosenescence, Boa constrictor, ageing, reptile, thymus, spleen, progressive fibrosis, inflammageing","lastPublishedDoi":"10.21203/rs.3.rs-6171887/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6171887/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAgeing is a complex biological process associated with the decline in immune function, known as immunosenescence, which leads to increased vulnerability to infections and other immune-related diseases. Immunosenescence is a focus of research in mammals and has been particularly well studied in laboratory rodents. However, whether the phenomenon is also a feature in poikilothermic animals such as reptiles, has not been investigated so far. This study explored the lymphoid tissue (spleen and thymus) of \u003cem\u003eBoa constrictor\u003c/em\u003e, a boid snake indigenous to South and Central America and Mexico, but widely kept in captivity all over the world, for potential age-related changes. We observed a significant decrease in cellularity in the spleen, coupled with an increase in organ size correlated with age. In both spleen and thymus the connective tissue of capsule and trabeculae increased significantly with age, indicative of progressive fibrosis. In addition, several changes were observed with increasing frequency in older animals, epithelial hyperplasia in the thymic medulla as well stromal fibrosis and an increasing infiltration by so-called granular cells in both organs. Granular cells likely represent a leukocyte subtype; their presence indicates a progressive chronic low-grade inflammatory state in the lymphoid organs, a feature known as inflammageing in other animal classes. They may also play a role in the progressive fibrosis of the connective tissue. The results provide first evidence of immunosenescence in \u003cem\u003eB. constrictor\u003c/em\u003e and indicate similarities in the underlying processes across animal classes.\u003c/p\u003e","manuscriptTitle":"Morphological evidence of immunosenescence in Boa constrictor?","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-10 07:09:56","doi":"10.21203/rs.3.rs-6171887/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-26T05:31:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-23T14:14:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-17T01:12:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"276184195731430826360052692679758979586","date":"2025-04-09T22:30:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"59898575523892560103712680543658838512","date":"2025-03-24T14:14:07+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-13T09:32:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-07T08:11:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-07T08:08:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"Immunity \u0026 Ageing","date":"2025-03-06T15:19:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"immunity-and-ageing","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"iage","sideBox":"Learn more about [Immunity \u0026 Ageing](http://immunityageing.biomedcentral.com/)","snPcode":"12979","submissionUrl":"https://submission.nature.com/new-submission/12979/3","title":"Immunity \u0026 Ageing","twitterHandle":"@ImmunAllergyBMC","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b81732a6-fdc1-48d3-b311-6801b4a01a81","owner":[],"postedDate":"March 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-23T15:59:59+00:00","versionOfRecord":{"articleIdentity":"rs-6171887","link":"https://doi.org/10.1186/s12979-025-00519-7","journal":{"identity":"immunity-and-ageing","isVorOnly":false,"title":"Immunity \u0026 Ageing"},"publishedOn":"2025-06-19 15:57:16","publishedOnDateReadable":"June 19th, 2025"},"versionCreatedAt":"2025-03-10 07:09:56","video":"","vorDoi":"10.1186/s12979-025-00519-7","vorDoiUrl":"https://doi.org/10.1186/s12979-025-00519-7","workflowStages":[]},"version":"v1","identity":"rs-6171887","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6171887","identity":"rs-6171887","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}