GENETIC ANALYSIS OF DEVELOPMENTAL MECHANISMS IN HYDRA I. SEXUAL REPRODUCTION OF HYDRA MAGNIPAPILLATA AND ISOLATION OF MUTANTS

Hydra magnipapillata strains collected from various localities in Japan were induced to reproduce sexually.
From the survival data of the progeny, it was calculated that H. magnipapillata contained an average of between 3.5 and 4.0 lethal equivalent units of recessive deleterious genes per gamate (between 7.0 and 8.0 per animal).
Various types of developmental mutants were found among the offspring of crosses made between strains isolated from the same ponds. The mutant types isolated included mini strains, maxi strains, multi-headed strains, nematocyst-deficient strains, regeneration-deficient strains and male sterile strains. The characters of these strains were stably transmissible to the successive progeny produced by budding. These strains therefore were propagated by budding and maintained as clonal lines to be used later for developmental studies.     

ANOMALOUS CONTRACTION OF INVERTEBRATE STRIATED MUSCLE

The phenomenon of A band shortening or contraction has been investigated in glycerinated myofibrils of Pecten irradians, Homarus americanus, Cambarus virilis, and Limulus polyphemus through the techniques of ultraviolet microbeam inactivation and polarization microscopy. With the former method, it has been shown that these muscles, even though exhibiting the shortening effect, contract in a manner consistent with only the sliding filament model. Intrinsic birefringence studies have indicated no significant changes in mass distribution or orientation within the shortened A bands. Except in the case of Limulus muscle, the shortening effect was seen only in contraction under tension. The magnitude of this anomalous phenomenon was dependent upon glycerination time and has been duplicated in rabbit psoas muscle through brief trypsin treatment. A band shortening could not be observed in glutaraldehyde-fixed muscle or in myofibrils glycerinated for only short periods. It has been concluded that the phenomenon of A band contraction is an artifact induced by the glycerination procedure, possibly through weakening of the sarcomere structure. However, the fact that the A band shortens under tension rather than lengthens poses an interesting paradox.     

EFFECTS OF COLCEMID ON HYDRA VIRIDIS WITH MULTIPLE-PEDUNCLE GRAFTS

The effect of Colcemid on multiple-graft Hydra viridis containing three peduncles (3p) grafted in tandem is reported. At grafting, the apical member of a 3p animal is a foot-less host, the middle member, an isolated peduncle, and the proximal member, a foot-bearing peduncle. In different experimental situations all three members are treated with Colcemid, the host member alone is treated, or the host member is not treated but the others are. Control animals have only non-treated members. Head and foot regeneration on graft pieces and the formation of waists and separations at the borders between these pieces are monitored and the significance of differences between frequencies tested by Chi square. Head formation on the middle and proximal peduncles is promoted by Colcemid when all pieces are treated, but animals with treated host pieces have significantly higher frequencies of heads on their non-treated proximal pieces than other experimental and control animals. Animals with Colcemid-treated proximal peduncles do not have significantly more heads on the most proximal peduncle compared to controls. Colcemid does not alter the frequency of foot formation significantly on the host peduncles of animals in the different categories, but foot formation is increased on the middle pieces of animals with three treated pieces and on animals with middle and proximal treated pieces. The rate of waist formation and graft separation is retarded when all three or just the host piece is treated, but not when only the middle and proximal pieces are treated. The direct action of Colcemid on nerves and on dividing cells may be responsible for these effects.     

水螅觸手數目之觀察

水螅(Hydra oligactus Pallus)之觸手數目,在天然環境中,可以有自十個至五個之差異。早期之研究皆以為觸手數目之决定皆由于水螅之大小及年齡。但實際觀察中有二點,顯示觸手數目似與水螅之大小無關:(1)此二者之間之相關係數極低,(2)水螅在生長條件最優時,可以增加其體積之大小,而觸手之數目不增多。但在某種環境下,水螅之觸手確能增加。此種環境之一即為華氏五十五度左右之温度。在該情形下,出芽生殖率降低,而觸手可以長出。此一情形,在自然間,即為晚晚春及早秋之温度。故水螅經年後,可以增加觸手之數目,亦可謂,觸手數目舆水螅之年齡有關。觸手增多無一定之規則。平常皆增加一至二觸手。但有時亦可增加一圈觸手(四個至五個)。水螅觸手數目正常或係六個,最少為三個,最多為十二個。但此二情形之水螅曾不能存在長久。三個觸手之水螅不久即長出第四個觸手,十二個觸手之水螅則不久即縱裂為二個水螅。     

THE STRUCTURAL ORGANIZATION OF THE SEPTATE AND GAP JUNCTIONS OF HYDRA

The septate junctions and gap junctions of Hydra were studied utilizing the extracellular tracers lanthanum hydroxide and ruthenium red. Analysis of the septate junction from four perspectives has shown that each septum consists of a single row of hexagons sharing common sides of 50–60 A. Each hexagon is folded into chair configuration. Two sets of projections emanate from the corners of the hexagons. One set (A projections) attaches the hexagons to the cell membranes at 80–100-A intervals, while the other set (V projections) joins some adjacent septa to each other. The septate junctions generally contain a few large interseptal spaces and a few septa which do not extend the full length of the junction. Basal to the septate junctions the cells in each layer are joined by numerous gap junctions. Gap junctions also join the muscular processes in each layer as well as those which connect the layers across the mesoglea. The gap junctions of Hydra are composed of rounded plaques 0.15–0.5 µ in diameter which contain 85-A hexagonally packed subunits. Each plaque is delimited from the surrounding intercellular space by a single 40-A band. Large numbers of these plaques are tightly packed, often lying about 20 A apart. This en plaque configuration of the gap junctions of Hydra contrasts with their sparser, more widely separated distribution in many vertebrate tissues. These studies conclude that the septate junction may possess some barrier properties and that both junctions are important in intercellular adhesion. On a morphological basis, the gap junction appears to be more suitable for intercellular coupling than the septate junction.     

STRUCTURE AND REPRODUCTION OF THE ALGAL SYMBIONTS OF HYDRA VIRIDIS1

The algal symbionts of Hydra viridis are found within vacuoles of the gastrodermal digestive cells of the host. Electron microscopy reveals that the symbionts possess cell walls, and that their reproductive cycle follows the general pattern of free-living Chlorella. Nuclear and chloroplast divisions arc followed by formation of new cell walls, the Golgi apparatus being quite active during cell wall synthesis. Autospores are released when the parent wall ruptures. The autospores are then usually segregated into separate animal vacuoles. Remnants of the ruptured parent wall persist in the vacuoles for an indefinite period. The ruptured parent walls curl at the breakage clefts, forming double-layered scroll-like structures. The fate of these wall remnants has not been firmly established. Long-term starvation of the animals does not result in a detectable change in the structure of the symbionts, and they continue to divide and to store carbohydrate as starch grains.     

ELEMENT RESPONSES OF AGAVES

To help identify possible element stresses, seedling responses of Agave deserti to high concentrations of various elements were examined by monitoring both 12-day growth in hydroponic solution and 6-month growth in sand culture. In addition, nocturnal acid accumulation by adult plants of six agave species was related to element levels in their chlorenchyma. Compared with common agronomic plants, seedlings of A. deserti were quite sensitive to salinity, with 50 mm NaCl greatly reducing root elongation in hydroponic solution and watering with 25 mm NaCl preventing growth in sand culture. The seedlings were rather insensitive to Ca concentrations from 0.2 to 5 mm and to pH from pH 5 to 8. They also tolerated high levels of B and of the heavy metals Cu and Zn. Nocturnal acid accumulation by adult plants of the six agave species was positively correlated with levels of 10 elements in the chlorenchyma, especially N (r² = 0. 70), B (r² = 0.51), and Ca (r² = 0.46). In contrast, nocturnal acid accumulation was weakly and negatively correlated with chlorenchyma Na (r² = 0.13), consistent with the deleterious effects of salinity on the growth of seedlings. Correlations between nocturnal acid accumulation and element content were consistent with previous fertilizer experiments with N, B, Ca, K, and P on A. sisalana. Element levels in the chlorenchyma of the six agave species were generally similar to those of previously studied cacti, including a low Na and high Ca level compared with agronomic plants.     

ROLE OF EDTA AND METALS IN MITOCHONDRIAL CONTRACTION

Studies comparing the state of hydration and dehydration of rat liver mitochondria to their content of ATP, Ca, and fatty acid, along with the rate of ATP hydrolysis, as well as microscopic appearance of mitochondria, have led to the following generalizations: 1. The competition between cationic translocations and water translocation for the available chemical energy (ATP) determines under many circumstances the water content of mitochondria. 2. Swelling of mitochondria by electron transport substrates is an example of the activation of the cationic translocations at the expense of water translocation. 3. Electron micrographic studies are interpreted to indicate that EDTA alone can cause condensation and dehydration of the mitochondrial matrix. However, both EDTA and substrate are necessary to remove appreciable quantities of water from mitochondrial intramembranous spaces. 4. Since the data in the accompanying report indicated that EDTA, in the absence of energy, decreased the permeability of mitochondrial membranes, it appears likely that ballooning of intramembranous spaces, following addition of EDTA, represents trapping of water between two semipermeable membranes following dehydration of mitochondrial matrix.     

A STUDY OF MYOFIBRIL SARCOMERE STRUCTURE DURING CONTRACTION

In the present investigation of cross-striated muscle fibers of axolotl, we succeeded in observing in one field of vision of the electron microscope all the stages of myofibril contraction. This allowed us to avoid errors in establishing the sequence of individual contraction stages. Our studies reveal a new contraction stage which appears at the shortening of the sarcomere below 74 per cent of the "resting length" but prior to the formation of typical "maximally shortened" sarcomeres, characterized by strong "contraction bands." At this stage, in the center of the sarcomere, at either side of the M line, a "secondary anisotropic" band arises which widens with further sarcomere contraction. At either side of this band, at the place of the former ("primary") anisotropic band, a "secondary isotropic" band is formed. A scheme of successive stages of contraction of the sarcomere is presented. The mechanisms of contraction for the first stage (from 100 to 79 per cent of the "resting length") and for the last stage (from 74 to 58 per cent of the "resting length") seem to be different. While the sliding of myofilaments with respect to one another can be assumed for the first stage, it is the spiralization of these structures which is the most likely explanation for the last stage. (An Abstract in German also appears at the end of this article.)     

PACEMAKER INTERACTION IN HYDRA

Previous reports have described in detail the characteristicsof two pacemaker systems in hydras. Each of these, the rhythmicpotential (RP) system located near the base of the polyp, andthe contraction burst (CB) system at the sub-hypostome, hasits own conducting system running the length of the animal,so that the impulses originated at either end spread throughoutthe organism. It has been postulated that these conducted impulsesof each system can reach and influence both the pacemakers ofits own, and those of the other, system. The over-all behaviorof the hydra would thus be the integrated responses of the effectorsresponding to these interdependent coordinating systems. Certain evidence favoring this hypothesis is presented. It isevident that there are interactions between the two systems,especially in the control of contraction burst initiation. Duringperiods in which RP impulses are appearing at short, regularintervals, contraction bursts are characteristically absent.Paradoxically when RP frequency is low, CB firing is also minimal.Normal CB activity levels are associated with intermediate RPfrequencies. There is also a frequency correlation between RP rate and theonset of individual contraction bursts. The interval betweenRP impulses is longer just before the first CB pulse than itis midway between bursts. It is shortest just following theburst. The longest RP intervals occur during contraction bursts.It appears that the initiation of endogenous CB activity isinfluenced by RP frequency in a manner comparable to the triggeringof the distinctive locomotor CB after RP pacemaker stimulation. Comparable work with other coelenterates has stressed the importanceboth of endogenous rhythmicity and ordered interaction betweenseparate pacemaker-driven coordinating systems. Hydras are notexceptional to this pattern, but they do show distinctive featurescompared to strictly sessile polyps. Further investigation maydisclose how the so-far unique RP pattern of repeated individualpulses and the absence of pulse bursts or trains is responsiblefor hydras' distinctive behavior.