Studies on the metabolism of the larval stages of hookworm
It has been recognized that ascaris eggs need oxygen for development(Brown 1928, Fairbairn 1957). Fairbairn(1957) found glycogen in the tissues of ascaris and measured it quantitatively.
Passey and Fairbairn(1955) reported that the developing ascaris eggs consumed much oxygen, whereas the matured eggs almost cased to respire and the amount of glycogen and lipids was decreased.
Kawazoe and his coworkers(1961) experienced that the content of glycogen in hatched larvae increased just after they migrated into the blood vessel and the blood sugar decreased reversely. This phenomenon may explain why the hatched larvae need to migrate into blood vessels for the supply of glycogen.
Fernando and Wong(1964) studied the glucose metabolism and glycogen synthesis of adult hookworms. However, there have been no reports concerning the oxygen consumption of migrating hookworm larvae yet.
In the present study, the author designed to measure the oxygen consumption and the amount of nutrition materials from rhabditoid to premature stage of hookworm in order to understand the reason for migration of hookworm larvae in the host.
Materials and Methods:
a) Free living stages; The hookworm(Ancylostoma caninum) larvae were divided into five groups according to the age of the culture. Culture(days) b) Tissue stage in abnormal host; 1,000 filariform larvae were given each to mouse orally. The mice were sacrificed at 24 and 48 hours after the infection, and the larvae from muscle, liver and lung were collected by the modified Baermann's method.
c) Preadult stage in proper host; Two dogs were infected with canine hookworm larvae 20,000 each, and the collected larvae from muscle, liver, lung and small intestine in 14 days after the infection were measured for respective purposes.
a) Body length: The larvae on the slide were killed by heating. 50 larvae were randomly sampled and the length measured with a micrometer following mathematic calculation, and ANOVA test was applied for significence.
b) Oxygen consumption: The oxygen consumption of hookworm larvae was measured by Warburg's standard manometric techniques (Umbreit, Burris & Stauffer, 1949).
c) Glycogen: Glycogen was isolated by the mothod of Good et al(1933) and glycogen was determined with anthrone reagent prepared according to Fales' method(1951). The optical densities were read in a Colemann spectrophotometer at 620 mμ.
d) Lipids: Total lipids was determined by the method of Folch et al(1951).
1) Free living stages; The length of the larvae was 452-479μ in rhabditoid stage and 596-647μ in filariform stage and no further development was observed. The amount of oxygen consumption was maximum, 919.7x10**-6 μl/hr./larva on the first
day after infection with rhabditoid larvae, and then gradually decreased according to the culture age reaching to 337.3x10**-6 μl/hr./larva, but there was no much difference between 6 days and 10 days culture larvae. The glycogen amount was the highest, 1411.29x10**-8mg/larva, on the 2nd day of the rhabditoid larvae, and decreased gradually. There was no difference between the 6th day and 10th day of the filariform larvae. The amount of total lipids was highest, 1956.4x10**-8 mg/larva on the 2nd day of the rhabditoid larvae and showed similar undulation with glycogen amount.
2) Tissue stages; The length of the larvae from host tissue after 24 and 48 hours of the infection was almost the same as those of the filariform stages. There were no significant differences of body length of the larvae which were collected from muscle, liver, and lung. However the oxygen consumption showed the highest value in the larvae from muscle 24 and 48 hours after the infection; 3892/6x10**-6μl/hr./larva and 8497.3x10**-6μl/hr/larva. The larvae from lung 24 and 48 hours after the infection showed the minimum value, 103.3
10**-6 μl/hr./larva and 145.0x10**-6μl/hr./larva. The glycogen amount in the larvae of 24 and 48 hours after the infection showed also similar tendencies. The larvae from the muscle showed 400.67x10**-8 mg/larva and 583.18x10**-8 mg/larva, and from the lungs were 134.22x10**-5mg/larva and 74.65x10**-8mg/larva. The total lipids from the larvae of 48 hours in various organs were as follows: 698.76x10**-8 mg/larva in muscle, 564.26x10**-8 mg/larva in liver, and 258.84x10**-8 mg/larva in lung.
3) Pre-adult stage: The average body length of the young adult of Ancylostoma caninum(14 days after the infection) collected from the small intestine of two dogs was 12.7mm; male 10.2mm, female 15.1mm. The length of the young adult was almost 20 times that of the filariform stage. The average glycogen amount of the worm was 27x-10**3mg/worm(15x10**-3mg/worm in male and 3.8x10**-3mg/worm in female) and the average amount of total lipids of the
worm was 0.77x10**-3 mg/worm (0.39x10**-3mg/worm in male and 1.15x10**-3 mg/worm in female). The oxygen consumption was 181.9 μl/hr./worm(125.4μl/hr./worm in male and 238.4 μl/hr./worm in femal)> which was as much as 600,0000 times that of the filariform larva.
Summary and Conclusion:
The body lengths, oxygen consumption rates, and the amounts of glycogen and lipids contents of free living and tissue migrating larvae of canine hookworm were examined. The following is a brief summary of the results:
1. The hatched larvae grew up rapidly until the reached to filariform stage, and significant manifestations of further development were observed neither in filariform stage nor tissue stages.
2. The developing rhabditoid larvae consummed much oxygen the filariform stage though the migrating larvae in muscle showed more than 10 times of oxygen consumption, compared with rhabditoid larvae and any other tissue stages.
3. The glycogen contents were the highest on the 2nd day rhabditoid larvae. The larvae from the muscle showed more less, though the amount was much higher than the value from liver and lung.
4. Lipids: The amount of total lipids was the highest on the 2nd day in rhabditoid larvae. The larvae from the muscle at 48 hours after the infection showed the highest amount of total lipids compared with from the liver and the lungs.
Through the above results, it is considered that the rhabditoid hookworm larvae deposit glycogen and lipids for development and activities during the free living stage, and the larvae migrate into the tissues restoring the nutrients for further development for the adult stage. 10 〃
Ⅴ 6 filariform
Ⅳ 4 〃
Ⅲ 2 rhabditoid
Ⅱ 1 Stage larvae