ZOOLOGICA SCIENTIFIC CONTRIBUTIONS OF THE NEW YORK ZOOLOGICAL SOCIETY VOLUME 54 • ISSUE 1 • SPRING, 1969 PUBLISHED BY THE SOCIETY The ZOOLOGICAL PARK, New York Contents PAGE 1. Laboratory Studies on Life-span, Growth, Aging, and Pathology of the Annual Fish, Cynolebias bellottii Steindachner. By Robert K. Liu and Roy L. Walford. Plates LIU; Text-figures 1-4 1 2. Direct Measurement of C02 Production During Flight in Small Birds. By John M. Teal. Text-figure 1 17 3. A Study of Experimentally Induced Endocytosis in a Teleost. I. Light Microscopy of Peripheral Blood Cell Responses. By Eva Lurie Weinreb and Stanley Weinreb. Plates I-III; Text-figure 1 25 Manuscripts must conform with Style Manual for Biological Journals (American Institute of Biological Sciences). All material must be typewritten, double-spaced. Erasable bond paper or mimeograph bond paper should not be used. Please submit an original and one copy of the manuscript. Zoologica is published quarterly by the New York Zoological Society at the New York Zoological Park. Bronx Park. Bronx, N. Y. 10460. and manuscripts, subscriptions, orders for back issues and changes of address should be sent to that address. Subscription rates: $6.00 per year; single numbers. $1.50. unless otherwise stated in the Society’s catalog of publications. Second-class postage paid at Bronx, N. Y. Published July 25, 1969 © 1969 New York Zoological Society. All rights reserved. 1 Laboratory Studies on Life-span, Growth, Aging, and Pathology of the Annual Fish, Cynolebias bellottii Steindachner1 Robert K. Liu2 and Roy L. Walford3 (Plates I-III; Text-figures 1-4) Various aspects of the biology of Cynolebias bellottii were investigated to provide base- line information necessary for studies on the physiology of aging. This species has proven to be a suitable animal for gerontological studies. Maintained since hatching at 22 ± 1°C., on an eight hour photoperiod with twice daily feedings of live, adult Artemia, males reached 50% mortality at 14.5 months, females at 15.5 months. Factors affecting the life-span of annual fishes are discussed. Growth was most rapid from hatching to two months, after which increases in length and weight continued up to ten months. Beyond these periods, individual fish may either continue to grow, show no increase in growth, or lose weight and/or decrease in length. Population averages for these parameters tended to decline with advanced age. Certain symptoms of pathology increased with age. Emacia- tion, equilibrium disturbances, and spinal curvature were the major gross symptoms. On the microscopic level, hyperplastic and changes were most frequent. Introduction A number of teleost fish are thought to be annuals (Myers, 1952). Such fish should be useful experimental animals for gerontologic research providing that their life- spans under laboratory conditions are as short or nearly as short as their designation “annual” implies, and that their husbandry is not overly difficult. We have shown that at 22°C. Cyno- lebias adloffi does indeed have a life-span of approximately one year, also that growth rate and life-span are significantly increased by lowering the ambient temperature (Walford and Liu, 1965; Liu and Walford, 1966). However, C. adloffi is prone to infections of Oodinium, piscine tuberculosis, and has a high incidence of reticuloendothelial hyperplasia. In the pres- ent report we present our initial observations with regard to mortality rates, growth rates throughout life, and gross and microscopic i Supported by United States Public Health Service Research Grant No. HD-00534. 2 Department of Zoology, University of California, Los Angeles, California 90024. aDepartment of Pathology, University of California School of Medicine, Los Angeles, California 90024. heterotopic thyroid tissue and gill filament pathology of the related annual, C. bellottii. This species is superior to C. adloffi as an animal for research on aging, in that it is much more resistant to infection than the latter and does not show a high incidence of idiopathic reticu- loendothelial hyperplasia. In addition, research on the biology of this species in semi-natural conditions (Bay, 1966, work in progress) pro- vides data for comparison against laboratory conditions. Our studies indicate that C. bellottii displays determinate growth and true senescence under the conditions maintained in our labora- tories. At 22°C., and under the feeding schedule specified, they displayed a 50% survival of 14.5 to 15.5 months, with a life table entirely charac- teristic of a population undergoing senescent change. No fish lived longer than 18 months. Material and Methods Adults were collected in the vicinity of Buenos Aires, Argentina, and sent to our labora- tory on 23 October 1963 by Professor Rogelio Lopez. Seven hundred and ninety eggs were col- lected at the end of two weeks when the last of 37 survivors died (19 males, 18 females out of the total shipment of 97 specimens). Five hun- 1 2 Zoologica: New York Zoological Society [54: 1 dred and sixty eggs were hatched at 22 ± 1°C. on 6 to 8 January 1964 and the viable juveniles were maintained at this temperature in an aqua- rium 33.0 cm wide x 88.9 cm long x 50.8 cm high, receiving eight hours of light daily. At one month, the population was counted and weighed; 35 individuals were selected as the experimental population and transferred to another tank of equal dimensions. At two months of age, the experimental population was counted, sexed, weighed, and placed in two tanks of the same size; 6 males and 1 1 females in one, and 6 males plus 12 females in the other. From three months onward, water was ex- changed monthly by means of a submerged pump between these separate tanks, in addition to removal and replacement of eight liters of water, in order to equalize conditions in both tanks and to reduce the concentration of growth- inhibiting factors (Rose, 1959a and 1959b; Kawamoto, 1961). For 21 months, the average pH was 5.2 (range 4. 0-7.0) and rarely differed by more than 0.4 between tanks. Except for procedures detailed in Walford and Liu ( 1965), no other means of regulating water quality were employed, since in its native habitat annual fish experience too diverse a range of conditions throughout the seasons (Boschi, 1957; Hoigne and Turner, pers. comm.) to be duplicated in the laboratory. Since the inception of our re- search on Cynolebias, we have encountered no problems with keeping these fish in either a mixture of tap and distilled water or entirely tap water (see Kinne, 1960 for an analysis of tap water from the Los Angeles Aqueduct). Fish were fed twice daily for 3 to 4 weeks on newly-hatched Artemia nauplii, then shifted gradually to a mixed diet of washed tubificid worms and live, adult brine shrimp for two more months, after which the diet was limited to the brine shrimp in order to reduce the possibility of infectious agents being introduced with the food. Enough Artemia were introduced daily after the third month to provide approximately one gram per wet weight for each fish. All techniques on collection and incubation of eggs, weighing and measuring, and autopsies and sectioning of histological material are given in Walford and Liu (1965). Results 1. Survival and life-span. A survival curve was calculated from the mortality data of Table I Table I Survival and Autopsy Data on Cynolebias bellottii MALES Time interval Number dying in months in interval Major gross and microscopic pathology 1-6 none 6-7 i 1 Culled 7-10 none 10-11 i 1 Emaciation. Heterotopic thyroid tissue. Mild reticuloendothelial hyperplasia. Hemosiderosis of spleen. 11-12 2 1 Slight emaciation. Heterotopic thyroid tissue. Chronic renal dis- ease with cyst formation. 2 Emaciation. Lesions around anus. Heterotopic thyroid tissue. Pharyngitis. 12-13 none 13-14 i 1 Emaciation. Equilibrium disturbance. Melanism. Neoplasm of gastrointestinal tract. Mild hyperplasia of pharyngeal thyroid tissue. 14-15 2 1 Emaciation. Extensive autolysis. Kidneys with severe vascular sclerosis. 2 Slight emaciation. Equilibrium disturbance. Lateral curvature of spine. Heterotopic thyroid tissue. Hemosiderosis of spleen. 15-16 1 1 Heterotopic thyroid tissue. Cystic degeneration of spleen. 16-17 3 1 Slight emaciation. Heterotopic thyroid tissue. Intracellular para- sitization of pancreas. 2 Equilibrium disturbance. Heterotopic thyroid tissue. Fatty de- generation of liver. Pharyngitis. 3 Equilibrium disturbance. Hyperplasia of pharyngeal thyroid tissue. 17-18 1 1 Equilibrium disturbance. Heterotopic thyroid tissue. Hyper- trophy of islet tissue of pancreas. Total 12 1969] Liu & Wolford: Laboratory Studies on Life-span, Growth, Aging, and Pathology of the Annual Fish, Cynobelias bellottii Steindacher 3 FEMALES Time interval Number dying in months in interval Major gross and microscopic pathology 1-3 none 3-4 i 1 Accidental death. 4-6 none 6-7 i 1 Culled 7-12 none 12-13 i 1 Equilibrium disturbance. Hemorrhage around anus. Mild inflam- mation of gill filaments. 13-14 2 1 Too autolyzed for histological analysis. 2 Extreme emaciation. Marked lordosis. Inflammed gastrointes- tinal tract. 14-15 8 1 Emaciation. Lordosis. Too autolyzed for histological analysis. 2 Emaciation. Marked lordosis. Degeneration of gill filaments. Inflamed gastrointestinal tract. 3 Inflammattion and hyperplasia of gill filaments. Inflammation of pharynx, kidneys and ovary. 4 Too autolyzed for histological analysis. 5 Too autolyzed for histological analysis. 6 Slight exophthalmia. Ovarian abscess. 7 Degeneration of gill filaments. Inclusion cysts in gut wall. 8 Emaciation. Slight inflammation of gill filaments. Atrophy of body musculature. Large hematopoietic tumor in abdominal cavity, with liver metases. Calcification of renal tubules. 15-16 3 1 Emaciation. Severe lordosis. Exophthalmia. 2 Slight emaciation. Inflammation and hyperplasia of gill filaments. Possible lymphocystis. Cystic degeneration of spleen. 3 Extreme emaciation. Lordosis. Exophthalmia. Degeneration of gill filaments. Fatty degeneration of liver. Granulomatous kidney and spleen. Concretions in lower gastrointestinal tract. 16-17 7 1 Too autolyzed for histological analysis. Total 23 2 Emaciation. Inflammation and hyperplasia of gill filaments. Concretions and inspissated gastrointestinal cysts. 3 Slight emaciation. Exophthalmia. Inflammation and hyperplasia of gill filaments. Islet cell tumor. Possible reticuloendothelial hyperpasia. 4 Slight lordosis. Inflammation and hyperplasia of gill filaments. Fatty degeneration of liver. 5 Emaciation. Lordosis. Inflammation and hyperplasia of gill filaments. 6 Slight lordosis. Too autolyzed for histological analysis. 7 Equilibrium disturbance. Slight lordosis. Heterotopic thyroid tissue. Degeneration of gill filaments. Fatty degeneration of spleen. by means of the equation of Leslie et al. ( 1955) which compensates for culls and accidental deaths (Text-fig. 1 ) . The form of this curve closely approximates the ideal survival curve of an experimental population exhibiting senes- cence and a fixed life-span (Comfort, 1961, 1964). For the males of our population, deaths occurred only after the tenth month, after which there was a rapid increase until the 50% mor- tality level was reached at approximately 14.5 months, with extinction at 18 months. A similar phenomenon occurred with females. Natural deaths began between 12 and 13 months of age, reached 50% at approximately 15.5 months with extinction at 17 months. No statistically significant sexual differences were found at either the midpoint or end of survival, although the first natural female mortality occurred two months later than that of the first male. 2. Growth. The monthly growth increments in length and weight are shown in Text-fig. 2. Growth is most rapid from hatching to two months, after which increases in length continue at a slower rate until about 10 months, while weight gain continues to six, seven, or ten months, depending upon the sex. Beyond these time periods, individual fish will often continue to grow, but the values of population samples will either fluctuate or decrease. Such fluctua- 4 Zoologica: New York Zoological Society [54: 1 Text-fig. 1. Survival curve for 10 male and 25 female Cynolebias bellottii maintained at 22 ± 1° C. throughout life. tions are usually due to small errors in measuring or to death of animals at either end of the meas- urement range. The aforementioned factors can contribute to a decreased average length, but such decreases largely reflect the development in aging animals of lordosis, kyphosis, and/or lateral curvature of the spine, thereby reducing the length measurement which is taken as the straight-line distance from the tip of the snout to the end of the caudal peduncle. Decreases in the mean weight with advancing age result from emaciation of individuals in the population samples. When present, emaciation usually oc- curred one to three months prior to death. None- theless, some individuals remained emaciated much longer than one to three months before dying (see Text-fig. 3; Plate I, figs. la-2b). It was possible because of their external appear- ance or behavior (see next section) to follow the growth of certain aging individuals. Only one of three males measured during the last months of its life-span showed a significant de- crease in weight; that same individual also de- creased in length due to lordosis (Table II). 3. Egg production. Eggs were collected from the third to the sixteenth month, but the very small number recovered each month suggested a labo- ratory artifact rather than decline in capability. One hundred eggs (4.3 eggs per female) were recovered during the third month, but thereafter only 0 to 1 egg was obtained per female, with a range of 3 to 25 total eggs per monthly sample. This small number contrasts markedly with the high egg production in certain stock tanks (790 eggs in nine days by a rapidly dying stock of 18 females, pers. obs., 1963), or by fish maintained in outdoor enclosures (1500 to 2000 eggs by three females in a two months period, Bay, 1966). 4. Aging and pathology. When each sample (10 of each sex, unless otherwise stated) was measured, symptoms of overt pathology were also looked for (Text-fig. 3). In brief, these were: equilibrium disturbance, so that the fish swims head down at an increasing angle from the horizontal or lies on the substrate unable to swim without sinking; extended branchioste- gals, caused by a swelling of the pharyngeal region internal to these structures; emaciation, evidenced by wasting first above the midlateral line and later along the abdomen (Plate I, fig. 2b); lordosis and kyphosis, a single or double curvature of the spine in the vertical plane; lateral curvature of the spine; exophthalmia; lesions on body; melanism. Although there was no way to determine whether these external signs were a direct result of physiologic aging, it can be said that these several types of overt pathology increased greatly in frequency as the population grew 1969] Liu & Walford: Laboratory Studies on Life-span, Growth, Aging, and Pathology of the Annual Fish, Cynobelias bellottii Steindacher 5 Text-fig. 2. Lengths and weights of males and females. At one month, population was not fully sexually differentiated, therefore the same observation on growth data is used for both sexes. The mean, two standard errors of the mean (2 80^ 5 70 K I 60 Vi 50 | | 40 <0 kj s 5 30 20 10 10 II 12 13 14 15 16 AGE (MONTHS) Text-fig. 3. The distribution of overt symptoms of pathology in relation to age. In both portions of the figure, the right ordinate is % in sample showing overt pathology, as indicated by the 8 symbols; the left ordinate is the actual number of survivors in the sample, as indicated by the survival curves. Liu & Walford: Laboratory Studies on Life-span, Growth, Aging, and Pathology of the 1969] Annual Fish, Cynobelias bellottii Steindacher 7 Table II Growth Data of Individual Males Month 12 Month 13 Month 14 Month 15 Animal number Length, Wt* Length, Wt. Length, Wt. Length, Wt. 1. 58.4; 4.20 58; 4.35 53f; 3.55 55; 3.25 2. 54; 1.95 54; 2.00 53f; 2.20 54; 2.20 3. 49; 2.55 49t; 2.50 49; 2.35 t difficulty in obtaining accurate length measurement, usually due to spinal curvature * length in mm, weight in gms fish died at 18 months of age. These time periods are longer than previously reported: Boschi ( 1957) cites approximately nine months as the life-span in nature, but more than a year in the laboratory; Myers (1952) less than one year in the field; Vaz-Ferreira, Sierra de Soriano and Scaglia de Paulete (1964) give 9 to 12 months as the life-span in nature. Boschi ( 1957 ) believes that a scarcity of food due to evapora- tion and reduction in volume of water in their natural habitats is the primary cause of death. Vaz-Ferreira et al. ( 1964) suggests reduction in water volume alone is the prime factor in mor- tality of the fish. Sternshein (1965) believes a diminishing food supply and high temperatures cause death. Thus, lack of food and physico- chemical changes in the environment, caused by the high temperatures of summer in the Southern Hemisphere have been postulated as the causes of death prior to the actual total des- sication of the habitat. Therefore, it has been difficult to determine whether under field con- ditions the fishes of the genus Cynolebias are obligate or facultative annuals. It is true that some species of Cynolebias are found occasion- ally in permanent bodies of water (Boschi, 1957; Hildemann and Walford, 1965) and it is known that eggs of C. bellottii can develop while kept continuously in water ( Scheel, 1 962 ) , nevertheless no observations are extant regard- ing life-span in the wild state in permanent water. Our own observations with C. adloffi, C. bel- lottii, and C. wolterstorffi (Walford and Liu, 1965; Liu and Walford, 1966; unpublished date) and that of Boschi (1957) show conclu- sively that these species can live much longer than a year in the laboratory. They can only be considered facultative annuals. A female C. adloffi kept at 16°., the last survivor of the ex- periment described in Liu and Walford (1966), died when 44 months old; populations of C. wolterstorffi maintained at the same temperature had only reached approximately 60% mortality at 30 months, and were still producing viable ova. It may well be that 16°C. is an optimal temperature (58 to 68°F. is indeed given as the best temperature range for C. bellottii by Boschi, 1957) not experienced for any long duration in the natural habitat of these fish. Life-span can also be prolonged by restricted feeding. In a pre- liminary experiment with C. bellottii kept at 23 ± 2°C., we fed them three times a week in- stead of the usual 13 to 14 times; the last male died at 24 months and the last female at 33 months of age. These results are similar to those obtained in rodents subjected to dietary restric- tion (McCay, 1952). Under more controlled ex- perimentation, it may be possible to show that a combination of high temperatures and de- creased food would decrease rather than in- crease mortality, as suggested by Boschi (1957) and Vaz-Ferreira et al. (1964). In addition to these environmental influences on life-span, certain aspects of the annual fish’s ontogeny may play important roles in the deter- mination of its total life-span. Although an egg can develop fully in two to three weeks, some eggs remain without sign of embryonic develop- ment for one to two years (maximum of 5J/2 years, Scheel, 1962). Tertiary developmental arrest can last over six months ( Wourms, 1965). Theoretically, therefore, an embryo can hatch after two to three weeks or after six years. Such variation may be inconsequential in nature (ex- cept as an adaptation to survival in case of pre- mature rains or extended drought) where each adult population has a life-span corresponding roughly to the availability of water, but in the laboratory, where eggs may be held for varying periods of time prior to hatching, differences in length of time spent as an embryo might sub- sequently affect life-span after hatching. Growth is one of the most easily monitored parameters that can be correlated with age. Nevertheless, few laboratory studies giving such data for the latter half of the life-span of fish have been conducted. It is not even fully known whether certain gross signs of aging, such as the fall in weight and length noted with the onset of senility in rodents and man, really occur to a comparable extent in fish. cull or accidental death Gross: emaciation 8 Zoologica: New York Zoological Society [54: 1 rJ o r4 on Tt i m cn vo < CU O n N N <3 5 o Pi o b O b u z b Pi D V u O b O 5 § D a b Pi c *5< o 3 cd >» OX) o O 3 O c/3 r- r/i C/D Uh « 0 o ^ c ^ X) 3 .S£ ZZ 'So co O 15 a -g e 5* E .3 . « u> oo jC -2 ’S3 £ g - ° Is •a “ '5 c u a> £ D. C/D .3 u a, " o £ o ° l— < 2 o bO •:=: w -r • - cd ’G Cl, yj 7D qj b S— /-S l_ * 8.1 g g s ’5) CO CO Cb >* a> o3 O u • — X 2 . Cb G 03 CO u. G G cd a _o 0X) >S 00 br x cd ° — i o OX) — - Id s 03 * E5 *o cl| g 5 S I I ° O w "3 ftS ox).s £ c/D 03 OX) £ O u cd u a c ? o « E .2 O E 2 6 '5- o o CO O o s K to 0 \0 O - OO £ OOOO^TfOO . -o -c> -o OUOU0 Y) d d ri tj- m +i +i ii +i +i +i +i oo O O so © oo O *o to so ri T) d oi ri OO NO O SO SC © o p p ri ’-h r- o I I I I I II n o o so © o o ^ O IT| h T O O >, © © to © ON o to ON 00 o o o o n so ri r- n to no +i +i +i ii +i +i +i oo O O so © O O to NO o o o p o h-miorrhOriON hlOhtOiONONOOO c H rq to rl | I O ~ oo ^ 3 - M *One blood sample available. 30 Zoologica: New York Zoological Society [54: 1 yama (1960) also referred to the mononuclear leukocytes in perch as transforming into macro- phages, epithelioid cells, and Langhans giant cells. Jordan (1938) considered the intermediate lymphocytes of teleosts as lymphoid hemoblasts, serving as stem cells. Reports on teleost hema- tology describing monocytes, large or intermedi- ate lymphocytes, and lymphoid hemoblasts, probably refer to transitional stages of similar cell types which may be considered, in most in- stances, to be lymphoid hemoblasts or lympho- blasts. Since most immature cell types (lymphoid hemoblasts, lymphoblasts, and intermediate stages) observed in this study were all closely related morphologically, these cells have been grouped together as blast cells. The responsive cell types noted in the goldfish, following injec- tion of the endocytic agent, were lymphocytes, immature and mature neutrophils, blast cells, and occasional eosinophils. The origin and func- tion of the polymorphonuclear leukocytes in tel- eosts have been described. Lymphoid hemo- blasts, originating from hemocytoblasts at hemo- poietic sites, differentiate into both leukocytes and erythrocytes. Neutrophils and eosinophils have been described as active phagocytes. The origin and function of mononuclear leukocytes, however, are less definitive. Responses of blast cells and immature neutrophils have not been extensively reported previously. Macrophage response was not noted in the fish studied. In the initial stages (first five hours) following injection, the neutrophil population consisted of immature stages comparable in maturation to mammalian promyelocytes, mye- locytes, and metamyelocytes. Their appearance was concomitant with the drop in mature lym- phocytes; however, no evidence of lymphocyte destruction was observed. The lymphocytes pres- ent were of the medium to large variety, and lymphoblasts were also prominent during this period. This blood picture reached a peak at six hours, after which the more usual picture of predominantly small lymphocytes and mature neutrophils was reestablished. Blast cell counts were variable because of the apparently rapid transformations occurring. Significant rises in blast cell populations were noted at three and six hours, coinciding with early lymphopenia and neutrophilia. The endocytic response was not typical of that seen in acute inflammatory reactions (also characterized by lymphopenia and neutrophilia) reported in a teleost (Wein- reb, 1958), since in endocytosis the predomi- nant cell types are both immature and mature polymorphonuclear and mononuclear leuko- cytes. Changes in the peripheral blood picture may be attributed to changes in production and out- put of cells from hemopioetic sites and/or changes in cell populations in the circulation. It seems more likely, because of the small numbers of hemoblasts and the lack of evidence of cell division, that cellular transformations or differ- entiations are occurring in the circulation. Previous reports related the mononuclear phagocytes to the hematological responses in fishes under various experimental conditions. Mesnil (1895) concluded, from a study of im- munological response in goldfish which had been intraperitoneally injected with Anthrax bacilli, that resistance was due to phagocytosis by mon- onuclear cells. Metchnikoff (1905) found that guinea pig erythrocytes were quickly phagocy- tosed by mononuclears following injection into goldfish. Intraperitoneally injected trypan blue was described in carp mononuclear phagocytes by Wislocki (1917). Mackmull and Michels ( 1932), in studies of colloidal carbon absorption from the peritoneal cavity of the cutter, Tauto- golabrus adspersus, found carbon in the poly- morphonuclear leukocytes and wandering pha- gocytes. Monocytes, difficult to distinguish from histiocytes, were reported as active phagocytes. Some phagocytic activity was also attributed to blood and tissue eosinophils. Katz (1950), in a study of the silver salmon fingerling’s response to bacterial infection, reported that neutrophils, lymphocytes, and monocytes were present, but only the mononuclear macrophages contained ingested bacteria. He considered the macrophage to be a specialized monocyte. Immature red cells, resembling basophilic normoblasts, were also present. Jakowska and Nigrelli (1953), in a study of bacteria infected guppies, noted phago- cytic eosinophils and macrophages at the sites of skin lesions. Watson (1961) noted macro- phages containing bacteria, neutrophils, and eosinophils at infection sites in goldfish. Both granulocytes and agranulocytes have been re- ported to be involved in fish blood cell responses, the macrophage which was prominent in bac- terial infection studies, was less important under conditions of aseptic stimulus. Mammalian studies indicate a more active role for the macrophage in both the normal and experimental animal. Rebuck et al. (1958) de- scribed transformation of lymphocytes into mac- rophages at acute inflammatory sites in man and noted that cells with features of the monocyte were observed prior to the appearance of the macrophage. Berman and Stulberg (1962), us- ing primary cultures of human macrophages, studied transformations of original cells in cul- tures where mitoses were practically absent. Their system provided an experimental mode) for studying interrelationships between macro- 1969] Weinreb & Weinreb: A Study of Experimentally Induced Endocytosis in a Teleost 31 phages and lymphocytes. They observed, in leu- kocyte cultures, that granulocytes degenerate and disappear, while lymphocytes and mono- cytes became hypertrophied and underwent a series of changes resulting in macrophage mor- phology. Berman and Stulberg concluded that development of macrophages in cultures oc- curred, in the absence of mitoses, by transfor- mation of lymphocytes, and probably small numbers of monocytes, of the original inocula. In a series of correlated morphological and bio- chemical studies on mammalian mononuclear phagocytes, Cohen et al. (1965a, 1965b, 1965c, 1966a, 1966b) have demonstrated, in both in vitro and in vivo systems, that monocyte-like cells differentiate into macrophages, and that endocytosis may be a major factor in macro- phage differentiation and function. The decrease in the number of small lympho- cytes, and the increase in medium and large lymphocytes, blast cells, and immature neutro- phils, suggest that there were cell-type trans- formations in the peripheral blood of the gold- fish under study. As noted, the number of herno- blasts, normally present, is too small to account for the large numbers of immature cells ob- served. The presence of blast cells, mainly lym- phoblasts, appears related to the altered lymphocyte picture. The multipotentiality of the lymphocyte has been extensively reported in both comparative and mammalian hematology. The function of the lymphocyte in the goldfish has not been precisely defined, yet this cell type constitutes over three-fourths of the circulating leukocytes. Its potential as a stem cell is con- sidered highly probable. It appears that changes in mononuclear cells are transient and reversible. The fate of neutro- phils, on the other hand, seems irreversible. The large hemoblast, prominent in the early reac- tions, is active in the phagocytic defense mecha- nism as well as serving as a precursor of cell types partaking in the reaction. The blast cells, mainly lymphoid hemoblasts, which are preva- lent during the first six hours following injection, are analogous to mammalian macrophages re- ported in other studies. It is possible that this cell is the result of “transformation” from the lymphocyte in the manner reported for mam- malian macrophage formation. The high incidence of blast cells in the cir- culation is attributed to the release from hemo- poietic sites and transition from other mono- nuclear cell types. The neutrophil, which is the most active phagocyte, may originate from the blast cell, via lymphocytic transformations, or the hemoblast. A similar sequence was suggested for human bone marrow cells based on morpho- logical and cytochemical studies of neutrophils (Ackerman, 1964). Hemopoietic activity of peripheral blood cells, such as lymphocytes, ap- pears to be stimulated by various stress condi- tions, including aseptic endocytosis. In the teleost, the less mature blood cells have the potential for phagocytic activity as well as cell proliferation and differentiation. Summary 1. The peripheral blood picture of the goldfish was studied to determine cell functions and analogies to mammalian cell types. Blood samples were collected at intervals, over a 26 hour period, from fish injected with Thoro- trast. Stained blood smears were examined for qualitative and quantitative changes. The mean ± S. E. and 95% range were deter- mined for all leukocytes. 2. Responsive elements were lymphocytes, im- mature and mature neutrophils, and a variety of blast cells. Lymphopenia and neutrophilia persisted for six hours. All neutrophilic ma- turation stages were present. The reaction de- creased after 12 hours, and the blood picture began to normalize after 1 8 hours. The rare macrophages did not respond. Mononuclear phagocytes were limited to lymphocytes and blast cells. 3. The lymphoid hemoblasts are analogous to mammalian macrophages studied under sim- ilar conditions. 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Three immature neu- trophils demonstrate stages in maturation comparable to mammalian early myelo- cyte (N1), later myelocyte (N2), and metamyelocyte (N3). A medium-size lymphocyte (L) is present. Fig. 7. A binucleate lymphocyte (L) and a ma- ture neutrophil (N) in a three hour sample. Fig. 8. A five hour sample demonstrating a small lymphoid hemoblast (B), with clear blue cytoplasm and extensive pseudopodia, a myelocytic neutrophil (N1), and a later metamyelocytic neutrophil (N2). Fig. 2. One hour sample containing lymphoblast (B), lymphocyte (L), and late myelo- cytic stage (N) in neutrophil development. Fig. 3. A large lymphoid hemoblast (B) showing extensive vacuolization (arrow), charac- teristic of blast cells noted in the three hour blood samples. Fig. 4. A large lymphoid hemoblast (B) and an immature neutrophil (N ) noted in a three hour sample. The blue-gray cytoplasm of the hemoblast closely resembles that of the mammalian monocyte. The neutro- phil, comparable to a metamyelocytic neutrophil in morphology, exhibits two types of granulation: larger, deeply acido- philic granules towards the cell periphery; and smaller, neutral granules in the pale “hof” area. Fig. 5. A lymphoblast (B) and examples of ery- throcyte maturation in a three hour blood sample. The erythrocytic stages are repre- sented by basophilic erythroblasts (E1), a polychromatic erythroblast (E2), and an erythrocyte (E3). Fig. 9. A five hour sample with an immature neutrophil, comparable to a late myelo- cytic stage, showing abundant fine granu- lation filling the cytoplasm. Fig. 10. A five hour sample demonstrating two metamyelocytic neutrophils. In addition to the fine granulation filling the cyto- plasm, there are vacuoles which may con- tain large, granular inclusions (arrows). Fig. 11. A late stage in lymphoblast development, in a six hour sample. This cell is smaller than less mature blast cells, with more condensed nuclear chromatin and deeper cytoplasmic basophilia. Fig. 12. Lymphocytes in a six hour sample are of small and medium variety. The medium lymphocyte shows cytoplasmic blebbing; the large vacuole (arrow) noted in one of the blebs contains amorphous, acidophilic material. Fig. 6. A dividing lymphocyte from a three hour blood sample. 34 Zooloeica: New York Zoological Society [54: 1 Fig. 13. Fig. 14. Fig. 15. Fig. 16. Fig. 17. Fig. 18. Plate III Lymphocyte vacuoles in a six hour sample contain amorphous, acidophilic material and small aggregates of particulate mat- ter (arrow). The cytoplasmic contour is irregular due to many small blebs. Early probasophilic stage of basophil maturation in an 18 hour sample. Large, very faintly stained granules fill the cyto- plasm. An intermediate stage in basophil devel- opment, from the preceding 18 hour sample, showing specific granulation. At this stage, unlike the mature cell, the granules do not fill the cytoplasm nor obliterate the nuclear outline. A mature basophil, from the same 18 hour blood sample, demonstrates the typical morphology of this stage. A transitional stage (B) between the late lymphoblast and early lymphocyte is typi- cal of mononuclear cells noted in the 24 hour blood samples. A small mature lym- phocyte (L) is also present. Three mature lymphocytes, demonstrating typical morphology, are representative of the cell populations noted in blood samples after 24 hours. WEINREB & WEINREB PLATE I A STUDY OF EXPERIMENTALLY INDUCED ENDOCYTOSIS IN A TELEOST. |. LIGHT MICROSCOPY OF PERIPHERAL BLOOD CELL RESPONSES WEINREB & WEINREB PLATE II A STUDY OF EXPERIMENTALLY INDUCED ENDOCYTOSIS IN A TELEOST. I. LIGHT MICROSCOPY OF PERIPHERAL BLOOD CELL RESPONSES WEINREB 8c WEINREB PLATE II! A STUDY OF EXPERIMENTALLY INDUCED ENDOCYTOSIS IN A TELEOST. |. LIGHT MICROSCOPY OF PERIPHERAL BLOOD CELL RESPONSES NEW YORK ZOOLOGICAL SOCIETY The Zoological Park, Bronx, N. Y. 10460 OFFICERS Fairfield Osborn Laurance S. Rockefeller Robert G. Goelet Chairman of the Board of Trustees President Executive Vice-President Henry Clay Frick, II Chairman °t the Executive Committee John Pierrepont Vice-President Howard Phipps, Jr. Treasurer Eben w Pyne Secretary Assistant Treasurer Edward R. Ricciuti Joan Van Haasteren Editor & Curator, Editorial Assistant Publications & Public Relations EDITORIAL COMMITTEE William G. Conway Donald R. Griffin Robert G. Goelet Chairman F. Wayne King Hugh B. House Peter R. Marler Ross F. Nigrelli William G. Conway General Director ZOOLOGICAL PARK William G. Conway . . . Director & Curator, Ornithology Hugh B. House .... Curator, Mammalogy Grace Davall . . Assistant Curator, Mammals & Birds Walter Auffenberg . . . Research Associate in Herpetology Joseph Bell . . Associate Curator, Ornithology F. Wayne King .... Curator, Herpetology Charles P. Gandal Veterinarian William Bridges . Curator of Publications Emeritus John M. Budinger . . . Consultant, Pathology Ben Sheffy Consultant, Nutrition James G. Doherty Mammalogist Donald F. Bruning Ornithologist Joseph A. Davis, Jr Scientific Assistant to the Director AQUARIUM Ross F. Nigrelli Director Robert A. Morris Curator Christopher W. Coates . . . Director Emeritus U. Erich Friese Assistant Curator Nixon Griffis .... Administrative Assistant Louis Mowbray . Research Associate in Field Biology OSBORN LABORATORIES OF MARINE SCIENCES Ross F. Nigrelli . . . Director and Pathologist Harry A. Charipper . . Research Associate in Martin F. Stempien, Jr. ... Assistant to the Histology Director & Bio-Organic Chemist Kenneth Gold Marine Ecologist George D. Ruggieri, S.J. . * . Coordinator of Myron Jacobs Neuroanatomist Research & Experimental Embryologist Klaus Kallman Fish Geneticist William Antopol . . . Research Associate in Vincent R. Liguori Microbiologist Comparative Pathology John J. A. McLaughlin . . Research Associate in C. M. Breder, Jr. ... Research Associate in Planktonology Ichthyology Martin P. Schreibman . . Research Associate in Jack T. Cecil Virologist Fish Endocrinology Jay Hyman Research Associate in Comparative Pathology INSTITUTE FOR RESEACH IN ANIMAL BEHAVIOR [Jointly operated by the Society and The Rockefeller University, and including the Society’s William Beebe Tropical Research Station, Trinidad, West Indies] Donald R. Griffin .... Director & Senior Fernando Nottebohm . . . Research Zoologist Research Zoologist George Schaller Research Zoologist Peter R. Marler . . . Senior Research Zoologist Thomas T. Struhsaker . . . Research Zoologist Jocelyn Crane . . . Senior Research Ethologist C. Alan Lill Resident Director Roger S. Payne Research Zoologist Q. Marcus Buchanan . . . Resident Director, Richard L. Penney .... Research Zoologist William Beebe Tropical Research Station Paul- Mundinger Research Associate