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[
Worm Breeder's Gazette,
1996]
As reported at the 1995 International Worm Meeting, our labs are generating a large number of transgenic C. elegans strains which have been transformed with cosmid DNA sequenced by the C.elegans Genome Sequencing Project (a joint U.S./U.K. effort headed by Drs. R. H. Waterston at
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[
Worm Breeder's Gazette,
1997]
Since our last report in WBG 14(2), we have expanded the cosmid transgenic library to include other chromosomes. Beyond the 147 cosmid trangenics we have constucted for chromosome III, we have now constructed transgenic strains for 61 cosmids from chromosome IV, 17 from chromosome X, 12 from chromosome I, 10 from chromosome II, and 6 from chromosome V. This brings the total number of cosmids available in transgenic strains to 253. The approximate location of these cosmids are shown as black bars on the figure below:
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[
Worm Breeder's Gazette,
1996]
The normal procedure for harvesting deficiency homozygotes for use in PCR is to allow a number of hermaphrodites, heterozyogous for a deficiency, to lay eggs on a clean plate for a short period of time, remove the hermaphrodites, and wait a sufficient period of time for non-arresting eggs to hatch. Eggs which do not hatch can usually be assumed be homozygous for the deficiency. As our lab utilizes this procedure often, it became tempting to sort deficiency homozygous eggs from their heterozygous siblings by moving eggs to a clean plate directly from the deficiency stock plate (using a platinum worm pick). This abridged procedure would save time and effort by eliminating the need to set up and remove large numbers of hermaphrodites. However, subsequent PCR testing of eggs which failed to hatch after being moved in this manner soon revealed something was amiss. Although wild-type eggs (and the deficiency heterozygotes in my experiment) normally hatch with relative certainty, it became apparent that something was occurring during the harvesting procedure which was resulting in unhatched heterozygotes. Our first hypothesis to explain this was that C. elegans eggs may go through a period of development when they are sensitive to being moved. To test this hypothesis, 100 wild-type (N2) adult hermaphrodites were set onto a plate and allowed to lay eggs for 2 hours before being removed. At every subsequent hour for 13 hours, 40 eggs were removed to a new plate using a well cooled platinum worm pick. The eggs were left at 20 C for 24 hours and scored for eggs which failed to hatch. The results were as follows: While the percentage of eggs which died at each time point generally decreased as the eggs got closer to hatching, there was still significant egg death throughout development. These results disprove the sensitivity period hypothesis (Note: We have not attempted to explain the periodicity suggested by our results, however we expect it is an artifact of our small sample size). Our second hypothesis was that eggs can be damaged by certain methods of transfer (specifically, by platinum worm pick). Again, wild-type hermaphrodites were set up and allowed to lay eggs. After four hours (the time point which resulted in the highest number of egg deaths in the previous experiment), 40 eggs were moved to a new plate using monofilament fishing line, and 40 eggs were moved by mouth pipetting the eggs directly into M9 buffer. In both cases all eggs hatched. These results suggest that eggs are sensitive to some characteristic of platinum worm picks which is not present in the other methods. The most obvious explanation is desiccation. The hydrophobic nature of the fishing line is perhaps prohibitive to the desiccation which occurs during transfer with a platinum pick. While these results are perhaps not unexpected, they provide a good reason for not departing too far from established protocols. At least, that is, when dealing with eggs.
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Gawne JM, Barbazuk BW, Stewart HI, Edgley ML, Vatcher GP, Franz NW, Ha TT, Baillie DL, Schein JE, Janke DL, O'Neil NJ, Rose AM
[
West Coast Worm Meeting,
1996]
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[
International C. elegans Meeting,
1997]
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[
International Worm Meeting,
2013]
Entomo-phoretic nematodes are not parasitic to insects but use insects for their transportation. Caenorhabditis japonica, a bacteria-feeding nematode, has a species-specific phoresy with a shield bug, Parastrachia japonensis, and its lifecycle is synchronized to the bug's life. The active dauer larvae (DL) show intensive host-seeking, high sensitivity to oxidative stress and less than 15 days of longevity without attaching the bug. On the other hand, quiescent DL on the bug survive more than 11 months. Thus, the quiescence associated with the bug seems an essential factor for nematode survivability. In the present study, survivabilities of quiescent and active DL were compared under several different conditions to examine the involvement of the bugs in nematode's longevity. Then transcripts and proteins were analyzed to compare gene expression between quiescent and active DL. The quiescent DL on the bug and active DL were kept in a container with 85% (dehydrated condition) or 97% (lightly dehydrated condition which immobilize the DL) of relative humidity (RH). The most active DL died in one week because of desiccation (85% RH) or fungal infection (97% RH). On the other hands, quiescent DL on the bug showed significantly higher survival rate under the same conditions. Further, the survivability of surface-sterilized active DL kept in 97% RH was almost the same as quiescent DL. Therefore, the bugs are likely to work as the shelter from dehydration, and are also providing anti-microbe activity to DL. The expressed genes and proteins also differed between quiescent and active DL. Expression of genes and proteins involved in several stress resistance, metabolic regulation and cuticle formation were significantly higher in quiescent DL. On the other hand, expression of genes and proteins involved in several metabolic related (activity regulation) were significantly higher in active DL. Our results suggest C. japonica DL use their host bug not only for transportation, but also as shelter from environmental stresses and microbe infection. Further, the quiescent stage-specific gene expression allows the extraordinary longevity of this stage.
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[
J Exp Biol,
2013]
Gravity on Earth is a constant stimulus and many organisms are able to perceive and respond to it. However, there is no clear evidence that nematodes respond to gravity. In this study, we demonstrated negative gravitaxis in a nematode using dauer larvae (DL) of Caenorhabditis japonica, which form an association with their carrier insect Parastrachia japonensis. Caenorhabditis japonica DL demonstrating nictation, a typical host-finding behavior, had a negative gravitactic behavior, whereas non-nictating C. japonica and C. elegans DL did not. The negative gravitactic index of nictating DL collected from younger nematode cultures was higher than that from older cultures. After a 24 h incubation in M9 buffer, nictating DL did not alter their negative gravitactic behavior, but a longer incubation resulted in less pronounced negative gravitaxis. These results are indicative of negative gravitaxis in nictating C. japonica DL, which is maintained once initiated, seems to be affected by the age of DL and does not appear to be a simple passive mechanism.
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[
International C. elegans Meeting,
1999]
Our lab has generated a cosmid transgenic library in order to rescue a number of essential genes on chromosome III (Janke et al 1997). We reported the discovery of a him (high incidence of males) phenotype exhibited by two separate transgenic lines containing either overlapping cosmids F56D2 or C05D2. We have isolated a single locus, C05D2.5 which when present on an extrachromosomal array results in a him phenotype. C05D2.5 has no significant homologies in the database but does have four cDNAs in Yuji Kohara's database indicating that C05D2.5 is an expressed gene. We are interested in discovering the role of this gene in chromosome segregation. Depending on the copy number of C05D2.5, between 4% and 27% self-progeny males are produced. In accordance with the increase in self-progeny males, there is an increase in embryonic lethality indicating autosomal death. We have also shown that nondisjunction occurs in the oocyte line and most probably the spermatocyte line. However, the transgenic progeny of a mating using a transgenic male and a Dpy-Unc hermaphrodite does not exhibit nondisjunction until the F2 stage indicating: 1) a build-up of the product is required and 2) there is not enough of the product in the sperm of the male passed on to the F1 progeny. Interestingly, this nondisjunction effect is carried on for at least three generations subsequent to losing the extrachromosomal array in transgenic animals. There is a decrease in male production and an increase in brood size with each successive generation. We have performed a RNAi experiment and found the result to be a significantly reduced nondisjunction effect of 4.1% males and a brood size of 166. We have been unable to detect any cytological defect in prophase of meiosis I, therefore the disruption in chromosome segregation appears to occur after diakinesis. In accordance with this we have found no disruption in recombination. As of yet we are unable to rule out either possibility of the overproduction of the RNA or protein product causing the effect. We are further characterizing the role of this novel gene in the process of chromosome segregation in C. elegans. Janke, D. L. et al. (1997) Interpreting a Sequenced Genome: Toward a Cosmid Transgenic Library of Caenorhabditis elegans. Genome Research 7974-985.
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[
Exp Gerontol,
2012]
The nematode dauer larva (DL) is a non-aging diapause stage. The DL of the model nematode Caenorhabditis elegans has been studied as a model system for aging and longevity. However, information on DL in other nematode species is limited. In this study, the survivorship, storage, energy consumption, and oxidative stress tolerance of Caenorhabditis japonica DL were examined. C. japonica is a close relative of C. elegans, but has species-specific phoretic associations with the shield bug Parastrachia japonensis. Also, its DL has a much longer lifespan than C. elegans in a biological setting. However, when C. japonica DLs were detached from their phoretic host, they did not survive more than 10 days while more than 80% of C. elegans survived under the same conditions. Also, C. japonica DL showed more active movement (swimming) and lower tolerance to oxidative stress than C. elegans DL. Because the concentration of triacylglycerol (TAG), the energy source of nematodes, did not decrease significantly during the experiment, exhaustion of the energy reservoir did not cause the low survivorship of C. japonica. Instead, low tolerance to oxidizing stress and increased production of reactive oxygen species in C. japonica were the main causes of the reduced survivorship. The fact that C. japonica DL cannot survive away from its insect host indicates that its longevity is increased by unknown factors derived from the host. Despite these significant differences between C. japonica and C. elegans, these two species are phylogenetically closely related (they are derived from a common ancestor). Therefore, C. japonica could be a good comparative system for C. elegans, and further physiological and molecular analyses of C. japonica DL may provide important information about the internal and external factors affecting the longevity of nematodes in general.