1 - 1 The Past Research on the Means of Enhancing the Self-Defense Function of Living Organisms: The past research and development effort and technological advances have failed to contain the worldwide spread of the deadly PRDV infection

The so-called "PRDV shock" has become the center-stage issue in the international ichthyo-pathological community giving rise to fervent research activities ranging from actual disease conditions, operational losses, preventative measures, all the way to the possibility of spread to other species. The Department of Applied Aquabiology of the National Fisheries University has played a leading role in developing effective PRDV countermeasures under the leadership of Dr. Yukinori Takahashi.

Obviously the most advanced function of homeostasis of living organisms is their immune mechanism. Though this function is peculiar to the more highly developed forms of life grouped as "Vertebrata," and absent in all forms of Invertebrata including shrimps and crabs, we nevertheless find these crustaceans are equipped with their own unique immune mechanism.

Professor Takahashi has been known as one of the foremost researchers of identifying chemicals extracted from natural substances with remarkable effect in enhancing PRDV-stricken shrimps' built-in self-defense function. The critical problem in this case, however, lies in the fact that a rigorous feeding schedule must be maintained with pellets treated with such chemicals, and then measures are yet to be found to the problem of decline in effectiveness after a period of repetitive use.

1 - 2 The Present Status of the Research & Development by the Tokyo Life Science Laboratory (TLSL) of a Unique Countermeasure Based on an Information Extraction/Transfer Technology

The TLSL's research and development activities date back to the early 1993 when the problem of PRDV control was first taken up by its laboratory located in Noto Peninsula, where a series of basic tests were conducted until a definitive conclusion was reached that there was enough scientific validity to continue with the effort.

From 1996 on, therefore, TLSL has continued with its effort to develop and perfect the system of countermeasures consisting of a specially designed powerful magnetic device, a special memory plate and a memory water containing fine metallic memory agent along with the necessary software for extracting from live organisms, and processing and transferring them for specific purposes. Actual aquaculture experiments have been conducted using laboratory ponds equipped with these devices to measure the effectiveness of this PRDV countermeasure.

While the conventional academic community as well as the leading shrimp farm operators remained indifferent at best, Professor Takahashi of the National Fisheries University has extended his full support to the TLSL research effort by providing both his own time and his university laboratory facilities for conducting the needed tests and experiments to them his own scientific and technical knowhows on such areas as determining the optimum strength of PRDV to be used for the survival ratio testing and the scientific control of the preparation of the test shrimps including their health conditions.

(1) Reversing the positive PRDV information

1) Recording the positive PRDV information and transferring the reversed (negative) information to memory water

And then, as a medium to transfer the information finally to shrimps, we treated the feed pellets with this (negative) information-laden memory water for feeding the shrimps. We also used ceramic balls which were baked by mixing clay with the same information-laden memory water, which were used for transmitting the information to the shrimp pond using a specially designed water circulation mechanism.

A series of tests using these two transmission media, pellets by way of the mouths of the shrimps and pond water environment itself transferring information through the skin of the shrimps, produced the average survival ratio of 20% to 40% for the tests in treated water and 5% to 10% for the tests in untreated water. Modest as they were, the tests nevertheless yielded a scientifically significant difference.

(2) Self-defense information transfer

1) Positive transfer of for enhancing self-defense function

2) The results of the tests

(3) Combining the "reversed" PRDV information and the positive information of enhanced self-defense capability

The survival ratio tests conducted with the combined method did produce a still more remarkable results, at the end of a 10-day test under attack by PRDV, of the shrimps fed with treated pellets yielding a ratio of 40% to 80% while only 0% to 10% of those fed with untreated pellets managed to survive, marking a convincingly significant difference.

(4) Conclusions and the salient points

As a result of the tests using the combined method above, it became evident that both the "reversed" PRDV information transfered through pond water and the positive information of enhanced self-defense capability transfered through feed pellets did successfully entered the shrimp bodies yielding even higher survival ratios than the sum of the two respective ratios derived from earlier separate tests.
This also meant that the information extracted from the blood corpuscles of the shrimps at the end of a 10-day period, during which they are continuously fed with pellets treated with the chemicals, did work as well by transfer via feed pellets.
The encouraging result was that the combination of these two different technologies did produce a remarkable synergistic effect.
Throughout the large number of survival ratio tests conducted during this period, it has been confirmed again and again that the life information extracted by TLSL's powerful magnetic device, which are in turn recorded in a special memory plate, reversed and repeatedly transferred through different memory devices/media did in fact end up in the bodies of the ailing shrimps producing the expected effect.

[2] The second group of experiments:

As a result of the first group of tests, it has been confirmed that PRDV can indeed by brought under control by feeding the virus-stricken shrimp with life information. Thus, during this period, our research effort focused on how the PRDV-related damages can best be contained and/or prevented using only the method of transferring information to pond water and cope with PRDV through the shrimps' living environment without using pellets as a medium.
If these experiments bring the expected results, it will signal the advent of a new operationally feasible countermeasure for the commercial shrimp farm operators to combat the PRDV threat.

(1) Direct transfer of information to shrimps by way of pond water

Admittedly, the sea water contains a variety of minerals and is considered capable of storing memories, though its level of memory capacity is considerably less than that of the memory water developed by the TLSL. Also, the sea water's effective length of memory retention is though to be much less than that of our memory water which is known by our tests to be at least 2 to 3 years. For this reason, therefore, we have developed the following device which enables us to pump information into the shrimp ponds on continuous basis.
In this test, we attached a powerful magnetic device to the outlet horse of the water circulation system using a special test pond, constantly pumping "reversed" PRDV information and enhanced positive self-defense information described in the foregoing pages into it. We used test ponds fully equipped in this fashion and observed such behaviors of shrimps as the survival ratio at the end of a 10-day period, sand bathing, skin casting and feeding habits while under attack by PRDV.

(2) The test results

Of the three test ponds, the fist one was being fed with the "reversed" information while under PRDV attack. The second one was being fed with no information while also under PRDV attack, and the third one contained healthy shrimps without inflow of information. The test results were that the survival ratio at the end of a 10-day period for the first was 60% to 75%, that of the second 5% to 10%, and that of the third 75% to 90%.
The reason why the healthy shrimps still suffered a nearly 20% loss was due to the stress created by the confined environment of the test pond. The first and the second tests were contrasty both in terms of survival ratio curve and feed consumption ratio curve, and obviously significant differences were observed in all tests conducted with varying levels in the force of magnetic device and strength of the PRDV concentrate. In all cases, therefore, the information fed into test ponds were transferred into shrimp bodies directly through skin to produce the expected results.

(3) Transfer of information to shrimps by way of pond water using aeration device

The above-described series of tests provided a confirmation of the fact that the sea water does memorize the information magnetically, there remained a practical problem of the sea water pumping system being operationally to costly.
Thus, the less expensive system of aerial transfer of information was developed, where aeration was used instead of circulating sea water. The first model failed, however, due to the fact that dry air cannot carry information from the magnetic device into the pond water. The second model was developed which uses the memory water sprayed into the air as it passes through the magnetic device to memorize and convey the information into the pond. This came to be called the "Wetair Method," and two series of tests were conducted with varying densities and quantities of memory water with successful results, but the further details are withheld due to the proprietary nature of the information.

(4) Illustrated test results

The experiments consist of test ponds, all under PRDV attack, fed with the two types of information discussed above carried by wet air into them. The "control (CONT)" pond is infected without feeding information while others are treated.

1) The 9th Attack Test

In this test conducted in 1998, an extremely potent PRDV concentrate, 100 thousand times stronger than what is enough to kill off all growing young shrimps, was used to forcibly infect the shrimps by skin contact.

At the end of the first 10 days, the survival ratios were from 66% to 80% for ponds A and B, while it was a disastrous 0% for the pond (CONT) left without feeding information, showing a rapid decline from 77% after the first day, 37% after two days, 5% after three days, and finally reaching 0% in five days.

2) Survival test under relatively weaker, but still powerful PRDV attack

With other conditions being the same, the potency of PRDV is reduced to about 10 instead of 100 thousand times as in the previous test.

The results at the end of the 10-day period were that the survival ratios for the treated ponds were 70% and 74% for A and B respectively while the ratio was 12% for the untreated pond (CONT).

3) Survival test with still weaker PRDV infection

With other conditions being the same, the potency of PRDV is reduced to about 1000 instead of 10 thousand times as in the second test. The results at the end of the 10-day period were that the survival ratio for the treated ponds was 93% for both A and B while the ratio was 53% for the untreated pond (CONT).

As a result of these tests, a scientifically significant difference in survival ratio has always been observed between the treated ponds and the untreated ponds (CONT). All these are specially designed rigorous tests to produce the expected results at the end of a 10-day period so that we can determine if there are significant differences between the treated and untreated ponds.

Thus, the survival ratios rendered do not necessarily indicate their applicability to the operational realities of commercial shrimp farms. There exist a complexity of causes for PRDV infection such as skin penetration, feeding on dead PRDV-stricken shrimps and other crustaceans living in the same environment like young crabs and their live fry, each shrimp pond having its own peculiar combinations of causes. One thing is for certain, however. Once infected, PRDV is bound to spread and destroy all shrimps in all stages of life within a time span of about two weeks.

1 - 3 Biological examination of the Process of Improving the Survival Ratio

It has become known, as a result of the tests conducted in the past, that fatal PRDV infection can occur without doubt through direct skin contact. Also, it has been observed that PRDV does penetrate into DNA and begins rapidly multiplying within several yours after initial infection.

The results of these tests indicate, however, that the combination of the "reversed" PRDV information and the enhanced self-defense information were in fact transferred from the magnetic device into pond water, and then allowed to affect PRDV by penetrating into the shrimp bodies through skin contact.

Upon administering PCR test on the shrimps still surviving after the 10-day tests, we found most of them carriers of PRDV. But, most interestingly, a small number of them were found negative non-carriers. It goes without saying that much more research as well as field tests are needed for scientifically explaining the mechanism, role and function of life information as used in this fashion. But, in the interim, the following efforts are currently under way from the biological as well as immunological and microbiological standpoints.

(1) Immunological examinations

Along with the second series of tests discussed in the foregoing pages, we have taken samples during the same test periods for conducting voracity tests and PO activities level tests.

1) Voracity ratios and indices

Next, we tested the devouring ratio which indicates the level of cell activities, which, in the case of
the untreated pond (CONT), remained rather constant throughout the 10-day test period at around 8% to 11%, while it increased from the 8% at the be ginning to 12-14% for the wet air circulation test and 8% to 22% for the direct transfer to the sea water of the pond.

The voracity indices with the actual number of cells in action taken into consideration increased, in the case of the untreated ponds (CONT),
from around 1.8 at the beginning of the test suddenly to a high 4.6 in the second day, and then, dropped down to the 2-1.5 level. In case of the ponds fed with information through wet air, after increasing up to around 4 during the first 6-day period (T1) to 2 at the end (T6), and then on, gradually dropped to 2.7 at (T10).
In the case of the ponds fed with information directly through sea water, the results were that an increase of around 5.3 during (T1) to (T3), about 4 at (T6), and then reached a high 10 at (T10). This means that the wet air tests registered an increase of from 10% to 100%, while the sea water tests from several tens of percentage points to a whopping 600% in index increase.

2) PO activities level

Shrimps also have the self-defense mechanism by means of which they internally produce quinone and melanin pigments with which to encircle and decimate alien subjects.

The polyphenol oxidase (PO) activities are usable as the indices of the production capability of these pigments. We have conducted our own examinations, along with the tests under 1), using time series for each of the test ponds using the light-absorption rate method.
In the case of the untreated ponds, it continued to drop from 0.2 at (T0) to (T3), and further down to 0.16 at (T10) showing no sign of increase. In the case of wet air method, after dropping from (T0) to (T3), it suddenly began rising from after (T6) reaching 3.5, a value 200% greater than that of the untreated ponds.
In the case of sea water direct transfer method, no change was observed up to (T3), but then, suddenly jumped from after (T6) to 4.5 and 5.5, amounting to 270% better performance than the untreated ponds.

(2) Microbiological tests

While PRDV can be kept in storage alive, it has not so far been possible to store and reproduce it in nutritive culture medium.
Therefore, we singled out vibrio penaeicida (vibrio in short in this paper), which has wrought havoc on Asia's shrimp farms, as our target, and conducted numerous series of tests to examine the effectiveness of positive and negative (="reversed") life information in controlling PRDV growth. A number of laboratory dishes shown in the chart contain PRDV-laden culture medium and placed so as to encircle the powerful magnetic device, which is loaded with a memory plate containing the positive life information extracted from PRDV.
N and S represent the opposite poles, and that part of the magnetic field which "reverse" the positive information is thought to exist not in the center region of the filed, but somewhere around the side rims. In fact, this the primary objective of this test was to determine where the "reversed" information are distributed around the magnetic device. In the white areas of the plates marked in the chart as "A" and the dotted areas marked as "B," no or little growth of PRDV was observed whereas in all other areas of the plates normal PRDV growth resulted, suggesting that these areas are where the positive information were "reversed."

This test called the 9th experiment was repeated more than dozen times, producing nearly identical result at all times. One of the remaining problems to be examined is the irregularity of the shape and size of the "no- and little-growth" areas.


Next, the memory plate containing the positive information was replaced by one with negative, or "reversed" PRDV information. The 10th experiment produced results which are almost nearly the exact opposite of the 9th experiment. As shown in the chart, the black areas around the side rims indicate the heavy PRDV growth whereas growth remained suppressed in all other areas.
We conducted the same experiment with larger number of plates covering the entire magnetic field. Similar tests were made over and below the position of the magnetic device. But, here again, they yielded complex shapes even though the general control pattern remained the same.

(3) Remaining tasks and challenges

To repeat what has already been said, we now consider that the validity of the life information extraction and transfer technology has been largely proven at least in its initial stage of research based on the results of the immunological and microbiological tests and experiments of the PRDV-stricken shrimps introduced in this paper.
In the last, we have conducted these tests and experiments more often as pilot projects at several shrimp farms in actual production, but at present we are also undertaking more rigorously scientific tests working with university research laboratories as well as technically more sophisticated experiments at shrimp farms in search of more scientific explanations and theoretical approaches. We are of the opinion that, in seeking fuller explanations, physics and chemistry will play a role as important as that of biology.

1 - 4 Using Test Tanks for developing practical technologies applicable to shrimp farms

The causes for PRDV infection are various, i.e., from parents to offsprings, from living environment through skin and/or feeding on the sick. We are yet to gain a definitive knowledge as to which are more dominant causes than others.

In the case of infection through feeding, one dead sick shrimp is eaten up by several live shrimps, which in turn will die in several to ten days. Thus, as this process spreads the disease in geometric progression. Also, infection through feeding results in a large amount of PRDV intake, which in turn makes the cycle so much shorter due to quicker death. Among the aforecited attack tests, the heaviest infection resulted in the near total decimation of the 95% of shrimps in just three days. We decided to recreate the same conditions in the laboratory test tanks in order to determine the effectiveness of our information technology as a PRDV countermeasure.

Also, in the case of infection through the living environment, the dead bodies of sick shrimps and crabs are spread across the bottom, making it unavoidable for PRDV to reach all corners during the 1-week period in which it remains actively infectious.

Prior to the release of shrimp fry, the pond is thoroughly disinfected by chlorination, but after release, strong disinfectant can no longer be used. Shrimp fry are being mass-produced to meet the needs of both shrimp farms and open sea release programs, but there are increasing suspicions being raised of vertical infection as well as horizontal disease transmission via the living environment. Therefore, our laboratory tank tests were also aimed at identifying effective methods of controlling PRDV infection during the process of mass-production of fry by means of "reversed" PRDV information transfer.

At present, various preventive measures are being tried by the shrimp farms including such chemicals as PG and fucoidan developed at the National Fisheries University, and it is our plan to seek out through these lab tank tests the best possible combination of the best of the traditional knowhows with our information transfer technology.

Here are the summation of these lab tank tests conducted so far.

(1) Controlling PRDV infection through feeding on dead sick shrimps

At commercial shrimp farms, it is no at all so easy to routinely collect dead shrimps day after day. Shrimps become active during the evening hours, and it is almost impossible to keep them away from feeding on dead and dying shrimp.
In this case, the test consisted of four tanks; one untreated tank received two PRDV-stricken shrimps mixed in with the healthy ones, which was left with one dying shrimp every morning in order to measure the rate of infection and death through feeding (CONT #1) while another untreated tank was left with all healthy shrimps (CONT #2). And then, the third and the fourth test tanks were treated with "reversed" PRDV information by wet air pumping while exposed to the same procedure of PRDV infection. In comparison with the untreated tanks, the other two tanks treated with "reverse" PRDV information produced a scientifically significant difference.

(2) Controlling PRDV infection by means of treating the shrimps with "reverse" PRDV information just prior to the virus attack

The transfer of the "reversed" PRDV information was allowed for one minute, and right after that, the shrimps were infected by PRDV through skin contact. One untreated tank was kept free from PRDV infection (CONT) while another tank was also kept PRDV-free but treated by wet air information transfer, the third tank was treated with "reversed" PRDV information without any treatment of the living environment, and the fourth tank the combination of the second and the third.
The results were that the fourth tank, the combination of the "reversed" PRDV information treatment and the wet air information transfer treatment of the living environment showed the highest rate of survival, followed by the third tank treated with "reversed" PRDV information and the second tank treated by wet air information transfer to the tank water.

(3) Controlling PRDV infection by combining PG and fucoidan with our information technology for greater effectiveness

The first tank was left alone with no treatment, and the second tanks was treated only with wet air information transfer, wile the third was the combination of wet air and PG treatment, the fourth tank the combination of wet air and fucoidan, and the fifth tank the combination of the third and the fourth.
The results were that the third tank with the combination of wet air and PG treatment registered the highest survival rate followed by the fourth with the combination of wet air and fucoidan, the fifth with the combination of all three, and finally the second only with wet air information transfer. And in each of these three cases, the survival rate had an obviously significant difference when compared to that of the first tank (CONT).

Judging from these test results above, we feel confident that we are nearer than ever to perfecting a truly operational technology which can be used commercially by shrimp farms. With this goal in mind, we are determined to delve into the more fundamental technological problems (to be discussed in Chapter Two) that must be solved for perfecting our information technology, while also aiming at integrating it with other cutting-edge scientific technologies.

Chapter Two -- Pilot Tests: Controlling the virus-caused shrimp diseases

2 - 1 Collaborating Shrimp Farms

During 1996, one overseas and one domestic shrimp farms collaborated with the TLSL test projects, one domestic farm during 1997, and from 1998 through 1999, four domestic shrimp farms have been working with the TLSL in the execution of the various tests and experiments discussed in this paper.

2 - 2 Test Methods

The wet air method was started in 1998, following the tests on pellet (sprayed with information-loaded memory water) and ceramic (to be spread on the pond floor with ceramic balls made of clay kneaded with information-carrying memory water) methods. During 1996, we experimented only with the "reversed" PRDV life information whereas in 1997, we began combining this with the enhanced self-defense information.

2 - 3 Summary of the Test Results

[1] Results of the overseas tests

The tests and experiments carried out in Malaysia during 1996 consisted only of feed pellets and ceramic balls methods containing the "reversed" PRDV life information. Working with a large-scale shrimp farm with ponds numbering in hundred and several tens raising black tiger shrimps. For a period of several years, however, this farm had produced only disappointingly small quantities of marketable shrimps under a severe attack in the early years of vibrio infection and later the onslaught of PRDV. As the latter continued to devastate the shrimp farms across the world, vibrio disease seems to have subsided. It remains true, however, these two are tending to combine to cause havoc in many cases.
In this experiment, we began treating the ponds in the above-described fashion from the stage right after the release of shrimp fry. The result was that all of the untreated ponds quickly developed symptoms of the PRDV-induced "white spot syndromes" resulting in the mass decimation of most shrimps. The subsequent fry release trials also ended up with disastrous results, leaving pitifully small quantities of marketable shrimps. On the other hand, all of the ten ponds treated by the TLSL "reversed" PRDV information witnessed a normal survival and growth records yielding more than 300 grams per square meter of water surface. Even though these ten ponds were scattered across the entire farm, making it difficult to collect reliable samples from each of them, the aggregate input and output figures seem to indicate that the treatment effect was fairly evenly spread.

[2] Results of the domestic tests

(1) The 1996 tests

Pellet and ceramic ball methods were tested in combination using only the "reversed" PRDV information at one of the shrimp farms on Shikoku Island (shrimp farm "A"). This farm continued to suffer serious damages, but, thanks to the TLSL technical cooperation, the farm succeeded to restore its production almost approaching the level of the planned target.

(2) The 1997 tests

Same type of tests continued at Shikoku Island's shrimp farm "A" again succeeding to approach the planned production target.

(3) The 1998 tests

1) Shikoku Island shrimp farm "A"

2) Shikoku Island shrimp farm "B"

3) Shikoku Island shrimp farm "C"

4) Shikoku Island shrimp farm "D"

5) PRDV-infected shrimps making full recovery carrying no virus at all

(4) The 1999 plans and what have taken place

We are conducting pilot tests and experiments at four different locations on the northern shores of Shikoku Island facing Seto Inland Sea. In addition to continuing with the shrimp farms "A" and "D" from the previous years, we have added farms "E" and "F." At "A" and "D," the season is in progress without a hitch, i.e., no major accidents, errors or disease outbreaks. At farms "E" and "F," which had been actually kept in fallow due to the violent past PRDV attacks, are now, as of August 31, production outlook is well over earlier expectations, and in-season shrimp shipments are already under way in order to provide more breathing room for the growing healthy shrimps.

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