Annual Fish Nothobranchius Guentheri: Morphological, Hematological and Biochemical Studies
【摘要】：The annual fish Redtail Notho, Nothobranchius guentheri has a laboratory lifespan of ~12 months and is becoming an emerging model for gaining insights into the aging mechanisms of vertebrates. Most current investigations on the fish involve developmental and age-related marker studies. Despite the popularity of N. guentheri in aging research, very little is known regarding physiological aging in the species. In the present study, therefore, an attempt was made to understand the physiological aspects of aging in the redtail Notho particularly its morphological growth indices, blood cell morphology, and biochemistry with a view to enrich the database for the species in general and in the broader context of aging research in particular.
The length-weight relation (LWR), Fulton‘s condition factor (K), and the relative condition factor (K_n) are some of the most widely used morphometric indices to assess fish condition. The condition or corpulence of fish is dependant upon the extent of energy reserves stored as fat or glycogen. Fish condition is known to vary in response to several extrinsic (mainly temperature and food availability) and intrinsic (maturity stage and sex) parameters. The first part of the present study dealt with the defining and understanding the growth indices for redtail Notho. Initially, the length and weight data of the species were recorded and analyzed under three lifespan periods (1, 2 and 3) each corresponding respectively to the first, second and the third four-month periods of the total life history of the fish. Given the highly sexually dimorphic nature of redtail Notho, the data were recorded separately for males and females under each age group. Three growth indices were computed from the data, i.e. (ⅰ) the exponent b of the LWR (W = aL~b), (ⅱ) Fulton‘s condition (K), and (ⅲ) the relative condition factor (K_n). In addition, the type of growth pattern for N. guentheri was assessed based on the mean b-value for the whole population taken together. The obtained growth indices were compared with reference to the median lifespan period (5–8 month-old). Analyses yielded some clear trends. The median lifespan occupied the median values for all the indices computed, the younger age group (up to 4 month-old) occupied the lowest and the oldest age group (9–12 month-old) the highest values. The mean length and weight for N. guentheri were found to be 2.9±0.8 cm and 0.371±0.314 g respectively. The mean Fulton‘s condition and relative condition factor were 1.21 0.8 and 0.98±0.16 respectively. Study of the distribution of length and weight across the different age groups revealed significant differences (p 0.05) as mean lengths and weights strongly tended to increase with age. The differential length–weight relations obtained for the various subsets of N. guentheri were all highly significant (p 0.001) with r~2 0.90. The exponent b was lowest for F1 (2.309) and highest for M2 (3.062) and had a mean value of 2.711 (S.E. 0.282). Shapiro-Wilk test did not reject the normality of distribution of b (W = 0.9181; p = 0.829). Thus, all the length– weight relations and the exponent b conformed to stringent statistical analyses.
The age factor appeared to have a pronounced influence on the growth indices of N. guentheri as a whole. Sex-based comparisons of the individuals showed the females to possess lower degrees of condition than their male counterparts did. The males of all age groups had a better condition than the females or even the combined population taken together. Pronounced sexual dimorphism in growth was evident, which was most conspicuous in terms of weight (2.15×) than in terms of length (1.28×). Sexual dimorphism in growth was especially more pronounced (p 0.05) during the first two growth phases (up to 4- and 5–8 months) and the differences became less significant in the older age group, especially in terms of length and weight (p 0.05). Sexually dimorphic differences, however, were still significant in terms of K and K_n in the older age group. This indicates that the females catch up with their male counterparts in terms of bodily dimensions (length–weight) later in the life.
Identification, characterization, and enumeration of the different blood cell types in the redtail Notho were conducted in the second part of the experiment. Given the great diversity of bony fishes, blood cells are known to vary with species but normally include erythrocytes (RBCs) and the leucocytes (WBCs) including thrombocytes, lymphocytes, monocytes, neutrophils (or heterophils), eosinophils, basophils, and some immature stages. Hematopoiesis in fishes is similar to that in mammals and occurs in two waves, primitive hematopoiesis and definitive hematopoiesis. The evaluation of basic hematology and blood chemistry parameters in fish is considered an important tool in assessing the health status as well as age-related changes. Blood was sampled from 3, 6, and 9 month-old males and females separately. Red (RBC) and white blood cell (WBC) counts were performed in triplicates using a hemacytometer. Natt-Herrick‘s blood diluent was used for the purpose. For differential leucocyte counting (DLC), two slides per fish were prepared and stained using the Wright–Giemsa (WG) stain and DLCs were performed by analyzing 30 random blood optical fields captured at 1000×from good quality blood smears. Finally, the cell types and counts were compared with the values obtained for the median lifespan (6 month-old) fish.
The erythrocytes (RBCs) were the most numerous of the cell types. The mean cell counts as well as the DLC did not differ significantly when compared to the median lifespan values at 6 months. The RBC and WBC counts for the species on a whole were 2.71±0.16 (×10~6μl~(–1)) and 1.28±0.14 (×10~3μl~(–1)) respectively. Accordingly, the DLC analysis revealed no differences either. The DLC for N. guentheri included thrombocytes (56.83±1.21%), monocytes and/or macrophages (14.66±0.73%), lymphocytes (13.08±0.31%), neutrophils (11.75±0.69%), and eosinophils (3.69±0.74%) as the prominent blood cell types. The presence of circulating macrophages was of a unique observation of the peripheral blood of the redtail Notho, and has been reported earlier in only a few teleosts studied so far.
Morphologically, the blood cell types were in general congruity with other teleosts. Both the mature and maturing (immature) erythrocytes were encountered. The mature forms were elliptical and more numerous than the immature forms, which tended to be more rounded and had a lower density than the mature ones. The nuclei occupied central or sub-central position. Though majority of the erythrocytes possessed a clear cytoplasm, reticulofilamentous particles were frequently encountered. Also observed occasionally were pyknocytic erythrocytes (with pointed tips), twin erythrocytes and/or special erythrocytes. The lymphocytes of redtail Notho were mostly rounded and sometime irregular in shape. The nuclei were dark and condensed and stained heavily in purple. The cytoplasm was very scant and stained in a light-blue hue. The monocytes were the largest blood cells encountered in N. guentheri and were mostly ovoid in shape with large nuclei that stained darker than the cytoplasm and occupied almost two-thirds of the cell area. The cytoplasm was uneven and showed the presence of granules. Some special macrophages were also encountered which possessed eosinophilic pink nucleus that often stained very lightly. The cytoplasm was hyaline. Eosinophils were mostly rounded and uncommon in smears. At times, cells with an irregular outline were also observed. Eosinophils were smaller than the neutrophils and had rounded nucleus. Neutrophils were rounded and possessed a horseshoe shaped and centrally placed nucleus. While the nucleus stained dark blue to purple, the cytoplasm stained in light purple and was found to be clear and transparent in most cases. Thrombocytes of redtail Notho displayed two different morphologies– elongated and rounded. Both forms possessed dark-blue stained nuclei with light-blue cytoplasm. The rounded or activated forms were abundant probably because no anti-coagulant was used. Overall, the monocytes and macrophages were the largest of the blood cells, while the lymphocytes and thrombocytes were the smallest.
In the third part of the study, a comprehensive biochemical profiling of the redtail Notho was carried out. Biochemical profiling is routinely practiced as a tool for health diagnosis in human and veterinary medicine, and has proven to be a valuable tool in analyzing the health status of farmed fish as well. Various biochemical indices provide reliable information on the health status, metabolic disorders, deficiencies and chronic stress status even before the clinical symptoms manifest. In this sense biochemistry tests are of a great prophylactic value for the ever-increasing fish culture sector.
Components of the comprehensive biochemistry panel are categorized as metabolites, proteins, enzymes, lipids and electrolytes. Metabolites include blood urea nitrogen (BUN), creatinine (CREA), uric acid (URIC), glucose (GLU), fructose (FRU) and bilirubins (BIL). Proteins include total proteins (TP), albumin (ALB), globulin (GLB), pre-albumin (PAB) and C-reactive protein (CRP). Enzymes include adenosine deaminase (ADA), alkaline phosphatase (ALP), alanine transferase (ALT), aspartate aminotransferase (AST),γ-glutamyl transferase (GGT), fucosidase (AFU), creatine kinase (CK), creatine kinase-MB (CKMB), lactate dehydrogenase (LDH), hydroxyl butyrate dehydrogenase (HBDH) and leucine amino peptidase (LAP). Lipids are represented by triglycerides (TRIG), cholesterol (CHOL), cholesterol high-density (HDL) and cholesterol low-density (LDL); electrolytes by calcium (CAL), chlorides (CLOR), potassium (POTA), magnesium (MAGN), sodium (SODM), phosphorous (PHOS). Biochemical profiles thus provide a comprehensive picture of the physiological health status or condition of an organism. Fishes are intimately linked to their aquatic environments and the physical environments in which fish live and the conditions governing them are known to influence their physiology in a profound manner. In this sense, biochemical profiles reflect the influence of a combination of extrinsic and intrinsic factors, including external stress, reproductive status, diseases, nutritional inadequacies and aging, and exposure to toxicants, which are known to significantly influence the blood chemistry.
In this study, a comprehensive biochemical profile for redtail Notho involving 40 analytes/analyte ratios (A/ARs) was obtained separately for the males and females at 3-, 6-, and 9- months of age using the whole body homogenates (WBHs) instead of serum. The reason why WBHs were used was due to the fact that the sizes of N. guentheri were small, making the blood collection difficult, in addition to the large sample volume requirements of the automatic analysis. Obtained data were normalized for osmolality (OSM) and only the normalized profiles were subject to further analyses. Age-related comparisons of the 3 and 9 month-old groups were performed with that of the median age group (6 month-old). While eleven and six analytes displayed decreasing and mixed age-based trends, only one analyte creatine kinase-MB (CKMB) showed increasing trend.
The decreasing trend in ALB was in accordance with the observed patterns in humans, primates and other vertebrates. However, some major trends were unique when compared to other vertebrates, notably, (ⅰ) the decreasing trends in the kidney metabolites (BUN and CREA), the lipids (CHOL and LDL), and the sugars (GLU and FRU); (ⅱ) the decreasing values of ALP and GLB; and (ⅲ) the increasing trends in the HDL with age. Apparently, no age-related tissue damage to the liver, the kidneys or the general musculature was evident from the tissue-specific profiles obtained. However, the inflammatory marker associated with the heart– CKMB showed an increasing trend with age. Thus, CKMB seems to be sensitive to aging changes in N. guentheri. Interestingly, most electrolytes with the exception of CLOR showed purely decreasing trends over age for N. guentheri. Although, the exact reasons could not be determined, these trends need further evaluation.
Overall, the data on decreasing or constant sugar and lipid contents with age indicates a calorie restriction-type situation in the later life of N. guentheri. Thus, older populations of N. guentheri seem to combat the ill effects aging by curbing the formation of advanced glycation endproducts (AGEs) modulated primarily through lowered or constant levels of GLU and FRU with increasing age. N. guentheri, thus, seem to have an effective inbuilt mechanism to overcome the excessive formation of the free radicals as also the glycosylated proteins thereby circumventing age-related stiffening of organs and tissues.
Biochemical profiles of the males and females differed significantly for HDL, CREA, URIC and CARB; males possessed higher values for these four analytes. Strikingly, the females had higher values for the lipids– CHOL and LDL, and for the bilirubins– TBIL and DBIL. Though these trends were not significant, they warrant further investigations. Noteworthy was the higher values for the inflammatory markers (CK and CKMB) for females, which seem to indicate that the females to experience muscular and cardiac dystrophy earlier than the males.
The cumulative biochemical profile of redtail Notho revealed both similarities and dissimilarities when compared with other teleosts and with the normal chemistry of humans. Given that the N. guentheri profiles were obtained from WBHs, such comparisons may not be completely valid since the profiles of other teleosts referred were obtained from serum. The use of WBHs may have influenced the biochemistry panel of redtail Notho, particularly with reference to their mineral composition. While the values of calcium and chloride were within the range reported for other teleosts, the magnesium, phosphorous and potassium levels were remarkably higher. The presence of skeletal components in the WBHs may have contributed to an increase in the mineral concentrations.
Exceptionally high levels of LDH values (8500 U/L) were recorded for N. guentheri, which were ~35 time the highest normal level for humans. However, higher levels of LDH are common with tissue samples than in serum. Higher LDH values, as such, are indicative of tissue breakdown and hemolysis, which given the fast developmental rate of N. guentheri, seem justified. Similarly, higher activity of ADA (1100U/L) was encountered, which is indicative of a higher nucleic acid turnover associated with fast growth in the species. As against a higher degree of ALP activity, no activity for ALT and AST was recorded. Use of WBHs could be the reason for this. However, were it to be an artifact of using WBHs, activities for most enzymes would have been altered, which was not the case. Thus, it is reasonable to assume that WBHs can provide a good representation of the analytes.
In summary, the annual fish Nothobranchius guentheri was investigated for the three parameters namely their morphological growth indices, their basic hematology and their comprehensive biochemical profile. Hematological and biochemical profiles of the median age group (6 month-old) were taken as the reference point for primary comparisons. The growth indices varied significantly (p 0.05) with age and with the gender with the median age group taking the median position for all indices tested (b, K, and K_n). Also, N. guentheri was found to follow the negative allometric growth pattern (b 3). The blood cell morphology of the redtail Notho was in accordance with other teleosts and the presence of circulating macrophages was of unique occurrence. Age-related trends in the biochemistry revealed some unique features and mostly hinted at gradual senescence in the species.
From an evolutionary perspective, the present findings are supportive of the indeterminate growth typical of fishes. It is widely acknowledged that the capacity of fish to keep growing beyond attainment of maturity confers upon them a continuing capacity of replacing old cells and delay senescence as compared to their mammalian and avian counterparts. Thus, there is an increased contribution of older age classes to fitness in fishes; with increasing fitness and size the egg-laying capacity increases. Added to this, the short life histories of killifishes place a high priority on the reproductive performance than other things combined resulting in an early setting and a long-lasting reproductive capability often lasting until death. Another point of view is that the sudden death syndrome is known to exist in some strains of killifishes that may make it difficult to detect any age-related changes. Taken together, all analyses point towards delayed or negligible senescence in N. guentheri up to 9 months of their age in terms of blood cell density and proportion, and biochemical profile, and over their entire lifespan in terms of growth.