Multicellular living organisms and unmodified parts thereof and – Nonhuman animal
Reexamination Certificate
2001-03-28
2002-06-18
Naff, David M. (Department: 1651)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
C424S405000, C435S260000, C435S283100, C435S307100
Reexamination Certificate
active
06407310
ABSTRACT:
TECHNICAL FIELD
The invention concerns the storage of entomopathogenic nematodes for transport or future use. More particularly it concerns the preparation for storage, and also the storage, of the third stage infective juveniles (commonly called “J3” nematodes) of nematodes belonging to the genera Steinernema (synonym Neoaplectana) and Heterorhabditis (synonym Chromonema), at controlled water activities.
BACKGROUND
It is well known that entomopathogenic nematodes in the families Steinernematidae and Heterorhabditidae have considerable potential for the biological control of a number of insect pests. Infective third stage juveniles (J3) of the nematodes (which can survive many weeks in the environment without feeding) are able to seek out an insect, penetrate into the insect's haemocoel and there release specific symbiotic bacteria (Xenorhabdus or Photorhabdus species). The bacteria kill the insect within a day or so and provide suitable conditions for the juvenile nematodes to progress to the adult stage, and for nematode reproduction.
The specification of International Patent Application No. PCT/AU93/00465, which was published as WIPO Publication No. WO 94/05150, contains a comprehensive summary of previously adopted methods for, and details of what was then recent work in, the storage of J3 entomopathogenic nematodes, including a description of an effective technique for the preparation for relatively long term storage of J3 nematodes. That technique involved mixing together an aqueous concentrate of clean J3 entomopathogenic nematodes and substantially anhydrous particles of a highly water-absorbent material, the proportions of the aqueous concentrate and water-absorbent material in the mixture being such that (a) sufficient water is absorbed from the concentrate by the absorbent particles to induce cryptobiosis of the nematodes, and (b) the mixture, after equilibrating, has a water activity in the range of from 0.80 to 0.995. Suitable arrangements for storing the cryptobiotic J3 nematodes at the selected water activity value are also described in WIPO Publication No. WO 94/05150.
DISCLOSURE OF THE PRESENT INVENTION
The first aspect of the present invention is an improvement in what has been, up to now, the preferred technique for preparing J3 entomopathogenic nematodes for storage, as described in WIPO Publication No. WO 94/05150, namely the selection of the water-absorbent material to be used in that method to induce cryptobiosis of the J3 nematodes, and to carry the J3 entomopathogenic nematodes that are in a state of induced cryptobiosis.
In WIPO Publication No. WO 94/05150, it is shown that polyacrylamide gel particles, or particles of starch polyacrylamide gel (optionally with polyacrylamide gel particles also present), or methyl cellulose powder, may be used as the medium both for use in the induction of the state of nematode cryptobiosis and for maintaining a selected water activity during subsequent storage.
In fact, methyl cellulose is not a highly water-absorbent compound and its recitation in WIPO Publication No. WO 94/05150 was inappropriate (being based on anecdotal evidence of methyl cellulose as a water absorbent). Methyl cellulose, which is used as an appetite suppressor, is a chemically derived compound which is soluble in water and which, in the course of dissolving, expands to form a glue-like product. Although anhydrous particles of methyl cellulose can absorb water from an aqueous cream of J3 entomopathogenic nematodes to induce cryptobiosis of the nematodes and establish a mixture that has the desired water activity, (a) the glue-like mass in which the J3 nematodes are distributed prevents air (oxygen) from reaching the nematodes, and (b) if, subsequently, water is added to the mixture of J3 nematodes, water and methyl cellulose, to release the nematodes for spraying, the methyl cellulose, being in solution, cannot be removed by sieving and the glue-like consistency of the mixture clogs the spray. nozzles. In fact, it is now clear that it is impractical to use anhydrous particles of methyl cellulose in the method described and claimed in WIPO Publication No. WO 94/05150.
Anhydrous particles of polyacrylamide gel, and of starch polyacrylamide gel, are significantly better as a storage substrate than attapulgite clay, which has been used previously for this purpose. This is primarily because, when particles of polyacrylamide gel, and of starch polyacrylamide gel, are used as described in WIPO Publication No. WO 94/05150, air is available to the nematodes while in their cryptobiotic state and it is not necessary to cool the nematodes during their storage. When attapulgite clay is used as the storage medium, a layered and compressed mixture of clay and cryptobiotic J3 nematodes is formed. This compressed mixture restricts the availability of air to the nematodes. This mixture, therefore, has to be kept in a refrigerator during storage to reduce the activity (and hence reduce the oxygen intake requirements) of the nematodes. However, the particles of polyacrylamide gel, or of starch polyacrylamide gel, retain a lot of water, particularly when a water activity of about 0.995 is established for the mixture. At this water activity level, particles of polyacrylamide gel, or of starch polyacrylamide gel, retain about 300 times their weight of water, which is really waste material.
There is one (quite old) recorded use of cellulose, in the form of a filter paper, as a medium on which J3 entomopathogenic nematodes can be stored. That disclosure is in the paper by J. F. Howell, entitled “New storage methods and improved trapping techniques for the parasitic nematode Neoaplectana carpocapsae”, which was published in the
Journal of Invertebrate Pathology
, Volume 33, pages 155 to 158, 1979. Howell put his J3 nematodes on a filter paper into a cold environment, thus ensuring that they did enter into a protected state, similar to cryptobiosis. However, there was no control of water activity and the J3 nematodes would not have survived if stored at a higher temperature. Moreover, storage of nematodes on a filter paper would not be a commercial proposition. If the nematodes should survive the storage period, they would be difficult to disperse in the field. The fibrous cellulose in a filter paper clogs up a spray nozzle, even when the filter paper is cut into small pieces. Presumably, to disperse the nematodes, they would have to be formed into a sprayable aqueous suspension by (i) immersing the filter papers containing the stored nematodes in water until the nematodes become active and leave the filter paper, then (ii) removing the filter papers from the dilute suspension of nematodes thus obtained, and (iii) optionally concentrating the suspension by decantation after allowing the nematodes to settle, or by collection of the nematodes using a muslin cloth, to form a sprayable suspension of J3 nematodes.
The present inventors have discovered that small particles (that is, particles having a size of less than 300 microns (300 &mgr;m), preferably less than 200 microns, more preferably less than about 100 microns, and most preferably less than 50 microns) of non-fibrous cellulose can be used as a storage medium for J3 entomopathogenic nematodes and that a mixture of J3 nematodes and such particulate cellulose, when suspended in water, is sprayable. The small particles of cellulose, unlike anhydrous particles of methyl cellulose, are not soluble. Cellulose particles are not highly water-absorbent (they absorb about 2.2 times their weight of water when establishing a water activity of about 0.995). When small anhydrous particles of cellulose are mixed with an aqueous cream of J3 entomopathogenic nematodes to induce cryptobiosis of the nematodes and establish a desired water activity, a fluffy mixture is produced, in which air (oxygen) is available to the J3 nematodes in the mixture. Thus the mixture (a) does not include a large amount of waste water (which is the case when particles of polyacrylamide gel, or of starch polyacrylamide gel, are used), and (b) does not
Bedding Robin Anthony
Butler Karen Louise
Clark Simone Daniela
Lacey Michael James
Naff David M.
Sughrue & Mion, PLLC
The Commonwealth of Australia Commonwealth Scientific and Indust
Ware Deborah K
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