Interacting Factors in the Control of the Crustacean Molt Cycle

In order to account for the known phenomena of the crustaceanmolt cycle, at least six factors must be postulated: a moltinghormone (20-OH-ecdysone), a molt-inhibiting hormone (MIH), ananecdysial limb autotomy factor, a proecdysial limb-autotomyfactor, a limb growth-inhibiting factor and an exuviation factor.Only the molting hormone and its derivatives have been chemicallywell defined. The various factors interact in complex ways tomaintain not only a coordinated proecdysial period in preparationfor exuviation but also a proecdysial period with the flexibilityto respond to such interim hazards as the loss of partiallyregenerated limbs.     

Development of Eimeria auburnensis in Cell Cultures

SYNOPSIS. Monolayer established cell line cultures of bovine kidney (Madin-Darby) and human intestine (Intestine 407), as well as embryonic bovine tracheal and embryonic spleen cell line cultures were inoculated with E. auburnensis sporozoites and observed for a maximum of 22 days. Mature 1st generation schizonts developed in the kidney, tracheal and spleen cells. In the intestine cells, trophozoites were seen in 3 of 4 experiments, but schizonts were not found. Sporozoites penetrated cells, beginning within a few minutes after inoculation. Penetration was usually accomplished within 10 seconds, and the body of the sporozoite underwent a slight constriction as it passed thru the host cell membrane. Some sporozoites left cells. Numerous intracellular sporozoites were observed in kidney, tracheal and spleen cultures. Crescent bodies were seen in the parasitophorous vacuole as early as 1 day after inoculation. At this time, the nuclei of most intracellular sporozoites had changed from vesicular to compact. Beginning 4 days after inoculation, enlarged sporozoites and parasites having a sporozoite shape, but with 2-5 nuclei, were frequently seen. These enlarged sporozoites and sporozoite-shaped schizonts evidently transformed into trophozoites and spheroidal schizonts by means of lateral outpocketings. Few trophozoites were seen. More immature schizonts developed in kidney cells than in the other cell types. The numbers of mature schizonts observed in kidney and tracheal cells were similar, but development occurred less consistently in the latter. Few immature and mature schizonts developed in spleen cells. Mature schizonts, first seen 9 days after inoculation, were considerably smaller than those reported from calves. Some motile merozoites were seen; evidently no development beyond these occurred. The nucleus and nucleolus of host cells were enlarged; this enlargement was not as pronounced as in infections in calves. Multiple host cell nuclei were frequently observed. Degenerative changes in the cultured cells and in the parasites usually occurred, beginning 9-17 days after inoculation; these were more pronounced in the spleen cells than in the others.     

Further Studies on in vitro Development of Eimeria bovis and Attempts to Obtain Second-Generation Schizonts*

SYNOPSIS. Eimeria bovis merozoites occurred in tissue culture medium removed from Leighton tube cultures of embryonic bovine tracheal cells beginning 12-14 days after inoculation with 270,000-369,000 sporozoites per tube. The number of merozoites produced in these cultures increased daily until a peak was reached 18-21 days after inoculation. In 3 experiments an average of 2.0–15.6 million merozoites per tube was produced during the 20-day observation period. When such merozoites were frozen in liquid nitrogen and stored 26–42 days, some were motile upon thawing. These merozoites as well as others freshly obtained from cell cultures and from calves were inoculated into 11 different types of cultured mammalian cells including primary, cell line and established cell line cultures. Some merozoites were exposed to substances normally found in the lumen of the gut, before or at the time of inoculation. Altho small numbers of intracellular merozoites were found, no further development was observed. Gametocytes were observed in the cecum of a calf 4 days after merozoites from cell cultures were introduced into a ligated cecum of the calf.     

Ultrastructural Study of Schizogony in Eimeria callospermophili*

SYNOPSIS The development of 1st generation schizonts of Eimeria callospermophili was studied with cell cultures and with experimentally infected host animals, Spermophilus armatus. Sporozoite-shaped schizonts each had 5-10 nuclei and all of the organelles of the sporozoite; each nucleus had a nucleolus and an associated Golgi apparatus. In stages immediately preceding merozoite formation, an intranuclear spindle apparatus with conical polar areas were observed near the outer margin of each nucleus. Two centrioles, each having 9 single peripheral tubules and one central tubule, were observed near each pole in some specimens. Merozoite formation began internally, with anlagen of 2 merozoites developing near each nucleus. The inner membrane of the merozoites first appeared as 2 dense thickenings adjacent to the polar cones and centrioles; subpellicular microtubules appeared simultaneously. Two anterior annuli and the conoid formed between the 2 thickenings. Vesicles, possibly of Golgi origin, were located next to the forming inner membrane. As the forming merozoites underwent elongation, a rhoptries anlage, a Golgi apparatus, refractile bodies, and mitochondria were incorporated into each. Sporozoite-shaped schizonts with merozoite anlagen transformed into spheroid or ovoid schizonts; at this time the conoid, rhoptries, micronemes, and the inner membrane of the pellicle gradually disappeared; several small refractile bodies were formed from the larger one. When development was about 1/3 complete, the immature merozoites began to grow outward from the surface of the schizont. In this phase of development, the single surface membrane of the schizont became the outer membrane of the merozoite's pellicle, and additional organelles, including the nucleus, were incorporated. Finally, the merozoites became pinched off, leaving a residual body. Development in cell cultures and host tissues was similar. This type of schizogony, previously undescribed in Eimeria, is compared with corresponding stages of development in other species of Eimeria and Sporozoa.     

DIDYMELLA STEM-ROT OF OUTDOOR TOMATOES

In studies on Didymella stem-rot of outdoor tomatoes several possible modes of introduction of the disease were investigated, together with methods of avoiding them. The pathogen was found to invest and penetrate the seeds in diseased fruit without destroying their viability and methods were devised of ridding the seeds of infection. It was not, however, possible to demonstrate the infection of mature plants from infected seed. It was found that the disease could be introduced on contaminated canes, in potting soil and from plant debris from the previous season's crop. The spread of the disease was encouraged by overhead irrigation.
Tests of strains of various species of Lycopersicon showed that one line of L. hirsutum was highly resistant to stem and root infection although another line was highly susceptible.     

Water Balance in the Land Crab, Gecarcinus lateralis, During the Intermolt Cycle

Gecarcinus lateralis can take moisture from a clamp substratumin amounts adequate for the needs of the entire intermolt cycle.It can also rehydrate in this way, even after severe dehydration. This crab is able to survive for many months when free waterof a wide range of salinities (0.30; = parts per thousand)is made available in a shallow dish. The crab dies within sevenweeks when the salinity of this water is 35. During proecdysisthe pericardial sacs of eyestalkless crabs become most swollenwhen the salinity of the available water is 15 or 23, and survivalduring and after ecdysis is greatest with water of 15. A crab in proecdysis shows no increase in the rate at whichwater enters following dehydration. Yet large amounts of waterare retained, particularly at the intermediate salinities. Maximalswelling of the pericardial sacs just prior to ecdysis is essentiallyequivalent in crabs with eyestalks, in eyestalkless crabs, andin eyestalkless crabs that have received an implant of centralnervous tissue. Hence, we conclude that a hormone causing theretention of water exists, but not in the eyestalks, in thebrain, or in the thoracic ganglionic mass. At ecdysis eyestalkless crabs show large increases in the dimensionsof the carapace, while crabs with eyestalks and eyestalklesscrabs that have received an implant of certain central nervoustissues show much less increase and may even show a decrease.Thus, we conclude that a hormone causing a release of waterat ecdysis is produced in the central nervous system. The advantages to the crab of a dual hormonal control of itswater balance are discussed.     

Eimeria scholtysecki n. sp. from Ord's Kangaroo Rat Dipodomys ordii*

SYNOPSIS. Eimeria scholtysecki n. sp. is described from Ord's kangaroo rat Dipodomys ordii. The sporulated oocysts are broadly ovoid to ellipsoid, averaging 24.6 by 19.6 μ. A polar granule is present. A micropyle and oocyst residuum are absent. The ovoid sporocysts average 12.1 by 8.0 μ, and have small, flattened Stieda bodies. The distinctive sporocyst residuum is composed of coarse granules. The mean prepatent period was 8.2 days. Five inoculated rats apparently became reinfected and discharged oocysts for 30 days or more.     

Sensitive detection of mycoplasmalike organisms in field-collected and in vitro propagated plants of Brassica, Hydrangea and Chrysanthemum by polymerase chain reaction

A polymerase chain reaction (PCR) protocol, previously designed for amplification of a DNA fragment from aster yellows mycoplasmalike organism (MLO), was employed to investigate the detection of MLO DNA in field-collected and in vitro micropropagated plants. PCR with template DNA extracted from symptomatic, naturally-infected samples of Brassica, Chrysanthemum and Hydrangea, each yielded a DNA band corresponding to 1.0 Kbp. However, no DNA product was observed when either infected Ranunculus (with phyllody disease) or Gladiolus with (symptoms of ‘germs fins’) was used as source of template nucleic acid for PCR; further experiments indicated absence of target DNA in the case of Ranunculus and the presence of substances in Gladiolus which inhibited the PCR. The MLO-specific DNA was detected by PCR using less than 95 pg of total nucleic acid (equivalent to total nucleic acid from 1.9, ug tissue) in the case of field-collected Hydrangea and less than 11.4 pg of nucleic acid (equivalent to total nucleic acid from 19 ng of tissue) in the case of field-collected Brassica. The findings illustrate highly sensitive detection of MLOs in both field-grown and in vitro micropropagated infected plants.     

Rapid laying by Brown-headed Cowbirds Molothrus ater and other parasitic birds

We compared the length of time parasitic and nonparasitic female birds spent on nests while laying eggs (laying bouts) to evaluate the hypothesis that rapid laving by parasitic Brown-headed Cowbirds Molothrus ater and other parasitic birds is a specialization for brood parasitism. Brown-headed Cowbirds typically spent less than 1 min on host nests while laying (41.0 ± 4.58 [mean ± s.e.] s, n = 21). In contrast, mean laving bouts of six nonparasitic icterine species ranged from 21.5 min to 53.4 min, and laying bouts of 13 other passerine species ranged from 20.7 min to 103.7 min. By spending only a few seconds on the nest while laying, brood parasites probably increase their chances of parasitizing nests unnoticed by hosts or, if noticed, are harassed by hosts for less time. Rapid laying may be adaptive if aggression by hosts can thwart attempted parasitism by chasing away the parasite, preventing the parasite from entering the nest or injuring the parasite. Rapid laying may increase the likelihood that the parasitic egg will be accepted. We tested some of these hypotheses by recording the responses of three frequently parasitized species to a stuffed female cowbird placed on their nests for 1 min. All species attacked the model vigorously; however, the mean time for discovery of the model ranged from 3 min to 17 min, ample time for female cowbirds to parasitize the nests. We concluded that rapid laying by parasitic birds is an adaptation for parasitism and, in Brown-headed Cowbirds, reduces the chances that the parasite will be attacked by hosts.