Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
  • A01K67/30—Rearing or breeding invertebrates

Definitions

• This invention concerns the storage for transport or future use of entomopathogenic nematodes. More particularly it concerns the storage and transport of the third stage infective juveniles of nematodes belonging to the genera Steinernema (synonym Neoaplectana) and Heterorhabditis (synonym Chromonema) using highly absorptive substances, such as polyacrylamide gels, at specific water activities.
• Nematodes have also been mass produced in liquid culture as detailed by G W Pace et al (see WIPO Publication No 86/01074), and by M J Friedman, S L Langston and S Pollitt (see the specification of International patent application No PCT/US88/04124, which is WIPO Publication No WO 89/04602).
• G W Pace et al see WIPO Publication No 86/01074
• M J Friedman, S L Langston and S Pollitt see the specification of International patent application No PCT/US88/04124, which is WIPO Publication No WO 89/04602.
• Popiel et al store their nematodes at relative humidities in the range of from 50 to 94 per cent, or from 95 to 99 per cent, while allowing for adequate oxygen supply. These humidities are maintained (i) with a hydrogel or fibrous matrix that has been impregnated with a saturated solution of potassium sulphate (which maintains a relative humidity of 97 per cent), (ii) using a saturated solution of potassium nitrate (which has a relative humidity of 94 per cent), or (iii) using solutions of sulphuric acid.
• the preferred technique involves storing the desiccated nematodes in a container in which there is also a saturated salt solution in a package made from a hydrophobic, vapour permeable membrane, such as GORETEX (trade mark) material.
• the cryptobiotic nematodes are stored in an airtight and moisture tight container with sufficient air space to accommodate the needs of the stored infective juveniles.
• Popiel et al claim that, using this technique, nematodes of the species Steinernema carpocapsae, Steinernema feltiae (synonym bibionis) and Heterorhabditis bacteriophora (synonym heliothidis) survived for several months with undiminished infectivity.
• nematodes In order to remove surplus water, a thin layer of nematodes is subjected to vacuum filtration followed by air drying. This takes much space and time. In addition, it is possible that nematodes may be excessively dried, particularly under commercial production conditions.
• the thin layer of nematodes must be removed from the treatment site and placed in containers for storage, with special provisions for maintaining the relative humidity near, but below, 100 per cent.
• a hydrogel structure made from a polysaccharide (such as agar, carrageenan or tragacanth), or a porous matrix of sponge, polyurethane foam or polyether foam, contains the nematodes, which "nictate" or “stand up and wiggle" on a relatively dry surface provided on the surface of the nematode-containing medium.
• the relative humidity of the air surrounding the nematode-containing medium is maintained at a value of at least 95 per cent by enclosing, with the medium, a quantity of "a water-liberating gel, such as swelled polyacrylamide".
• R A Bedding showed how clay (particularly attapulgite clay) can be used to store entomopathogenic nematodes, either by forming a homogenous mixture of nematodes with the clay (which is not a highly absorbent material) or by making a sandwich consisting of a layer of nematode cream between two layers of clay.
• This method (which is currently used commercially) is believed to owe its success to three major factors, namely (i) the nematodes are constrained from moving (thus conserving food reserves), (ii) perhaps, the clay adsorbs potentially toxic nematode excretory products, and (iii) by exposing nematodes to humidities of less than 100 per cent, preferably near to a water activity of 0.97, they are induced into the cryptobiotic state.
• a range of entomopathogenic nematode species can be stored in this way using either chips of calcined attapulgite clay or coarsely milled, calcined attapulgite clay.
• More than half the weight of the stored nematode product is clay, which adds to transport costs when air freight is used.
• This objective is achieved by using highly absorptive (but not necessarily adsorptive) gels, or other highly absorptive substances, to absorb surface water from an aqueous cream of nematodes and, having done this, to accurately provide a water activity within a narrow range suitable for inducing cryptobiosis of the nematodes without adversely affecting their survival.
• Water activity is defined as the ratio RH/100, where RH would be the relative humidity of the surrounding atmosphere in a sealed system.
• highly absorptive substance will be understood by persons who work with absorptive materials and are familiar with their classification. Without limiting the general understanding of the term “highly absorptive”, the preferred highly water-absorptive substances of the present invention are substances which absorb at least 75 per cent of their own weight of water to have a water activity in the range of from 0.80 to 0.995
• anhydrous or nearly anhydrous absorbent particles are preferably mixed with a nematode cream in predetermined combinations so that both the absorption of the surface (free) water and the establishment of the desired water activity are achieved without requiring further adjustment of the resultant mixture (for example, the addition of extra water).
• Fungicides and/or antibiotics may be mixed with the nematode cream or with the absorbent prior to mixing the nematode cream and absorbent together.
• the benefits of using highly absorbent substances are believed to be five-fold. Firstly, they remove surface water rapidly so that the nematodes form a "foam" within a few minutes. Where particles of an absorbent (such as polyacrylamide gel) that swells in the presence of water are used, large interstitial spaces are formed between the swollen particles. These spaces provide room for nematode foaming and for the better diffusion of gases. Thus an aerated matrix is rapidly produced and the nematodes suffer the effects of anaerobic conditions for a short time only.
• an absorbent such as polyacrylamide gel
• the mixture not only induces cryptobiosis in the nematodes, but it also, thereafter, maintains the water activity at a value which is appropriate for the continual storage of the nematodes.
• the weight of absorbent used can be significantly less than that of the nematode cream.
• Nematodes stored in this manner using highly absorbent substances can be reactivated by dispersing the nematodes and absorbent in water. However, they are most effectively reactivated by adding water to the nematode/absorbent combination so that the water activity exceeds 0.995 but remains below 1.00; the nematode/absorbent combination being then left for two to four hours before finally dispersing the nematodes and absorbent in a surplus of water. Where particles of the absorbent swell in the presence of water (for example, when the absorbent is polyacrylamide gel), it is then a simple matter to sieve out those particles to leave a clean suspension of infective juvenile nematodes.
• a method of storing third stage infective juveniles (J3) of entomopathogenic nematodes comprises forming a mixture of an aqueous concentrate (cream) of clean J3 nematodes and particles of a highly water-absorbent (as defined above) material, the proportions of the aqueous concentrate and water-absorbent material being such that the mixture, after equilibrating, has a water activity in the range of from 0.92 to 0.995.
• the water activity of the mixture is from 0.92 to 0.995, and most preferably between 0.95 and 0.99.
• the final value of the water activity of the mixture may take from 24 to 72 hours to establish.
• the absorbent may comprise from about 15 per cent to 75 per cent of the resulting mixture.
• experiments should be conducted to determine the water activity resulting from various combinations of it with nematode cream.
• the water content of the nematode cream must first be standardised. This can be achieved by making the number of clean infective juvenile nematodes per gram constant for any particular species of nematode or by standardising the viscosity of the nematode cream used. The more viscous the nematode cream, the lower the quantity of absorbent that is required to achieve a particular water activity of the final absorbent/nematodes mixture.
• the exact water activity required to give optimal induction of cryptobiosis varies a little from species to species of entomopathogenic nematodes. After a period of induction (usually 2 to 3 days), the nematodes may be stored with the mixture having the attained water activity, or the water activity of the resulting combination may be lowered by adding further absorbent.
• PAGs particulate anhydrous polyacrylamide gels
• a PAG having an individual dry particle weight of from 0.005 gm to 0.02 gm is preferred because, after swelling, such particles provide adequate interstitial space for nematode foaming and gaseous diffusion.
• Figure 1 is a graph showing the water activity of various mixtures of polyacrylamide gel and water.
• Figure 2 is graphical presentation of mortality data for various absorbent/nematodes combinations of the nematode species Steinernema carpocapsae.
• Figure 3 is a graphical presentation of the average dry weight of nematodes (S. carpocapsae) in stored samples after different storage times.
• entomopathogenic J3 nematodes that have been reared in a liquid culture frequently store less well than those reared in a solid culture (this is particularly true of Heterorhabditis species).
• utilisation by the nematodes of their internal food reserves prior to storing should be reduced to a minimum.
• nematodes used in the series of trials conducted by the present inventors to confirm the efficacy of the present invention were reared and extracted using the methods described by R A Bedding, M S Stanfield and G W Crompton in the specification of International patent application No PCT/AU91/00136 (WIPO Publication No WO 91/15569).
• the nematodes may be reared on insects in vivo or in liquid culture, provided the nematodes are free from appreciable amounts of extraneous matter remaining from the culture medium and are relatively free from nematode stages other than J3 (preferably no adult nematodes are present and certainly no more than 2 per cent of the nematodes should be adults).
• the resulting cream of nematodes contained from 0.5 to 3.5 million J3 nematodes per gram, depending upon the species involved and the amount of inter-nematode water remaining.
• various antifungal and antibiotic agents were added to and mixed with the sedimented nematodes while they were still in the tanks. Hence, after removal of much of the surface water (and thus also of most of the antibiotic and/or antifungal agent), some of the antibiotic and/or antifungal agent remained to be absorbed by the absorbent in the storage process.
• the antifungal agents used by the present inventors were humic acid, brown coal dust (which contains up to about 50 per cent humic acid), powdered sulphur, sulphurous acid, and a mixture of sulphurous acid and powdered sulphur.
• the technique usually adopted for combining the nematodes and the absorbent material was as follows. Particles of the absorbent were weighed and added to the appropriate weight of nematodes. (The appropriate weights were determined by prior experimentation to ascertain which combinations fall within the required range of water activities). The absorbent and nematodes were then immediately stirred and mixed together so that the absorbent particles were evenly distributed in the mixture.
• the water activity, of the mixture of nematodes and highly water-absorbent particles has to be in the range of from 0.80 to 0.995.
• the water activity is preferably in the range of from 0.92 to 0.995, and most preferably is in the range of from 0.95 to 0.99.
• polyacrylamide gel is the preferred absorbent and Figure 1 shows the ratio of water to polyacrylamide gel to achieve a water activity for the mixture in the range of from 0.95 to 0.995.
• the required water activity of the mixture is not attained immediately.
• the absorbent particles quickly take up the free surface water of the nematode cream, and then absorb water that is released from within the nematodes.
• the absorbent material has a water activity lower than its final value, which is attained after 24 to 72 hours.
• the water activity of the gel particles is 0.92 for the first four hours after the mixing has taken place. The water activity then increases to 0.94 eight hours after mixing, but does not attain the (intended) value of 0.97 until 24 hours after the mixing of the nematode cream and the absorbent particles.
• the containers were stored at the extended storage temperature of the experiment immediately after receiving a sample of the mixture of nematodes and absorbent material. In other experiments, the containers were stored firstly at 15°C for three days and then at the extended storage temperature. In all experiments, the extended storage was effected in a manner such that the water activity of the absorbent/nematodes combination was maintained at a value in the range of from 0.80 to 0.99.
• the squeezed cream had a viscosity such that when it was placed in a 4 cm open ended tube held vertically, it did not drop out of the tube within 2 minutes. (The addition of even small quantities of water to some of this cream reduced its viscosity so that it readily dropped from the tube within a few seconds.
• the squeezed cream was used to determine what water activities were achieved with various combinations of sieved polyacrylamide gel crystals (the crystals having an average mass of 0.01 gm) and this species of nematode, and how these water activities affected nematode mortality if the combination is stored at 23°C.
• the nematode cream was divided into seven batches and combined with the PAG crystals in the following proportions by weight:
• Nematodes were produced and processed as in Example 1, but with 10% more fat added to the rearing medium. Batches of from 200 gm to 500 gm of an aqueous cream of nematodes were combined with particles of sieved PAG (average particle weight, 0.01 gm). Surface water in the nematode cream could not be standardised as readily as in Example 1 because Heterorhabditis bacteriophora cream loses water much more readily than Steinernema carpocapsae. Accordingly, the H. bacteriophora nematodes were standardised by making a thick cream and then diluting it with water, so that there were about 2.5 million infective third stage juvenile nematodes per gram. This diluted cream of nematodes was combined with polyacrylamide gel particles as in Example 1.

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• 1993
  • 1993-09-10 CA CA002144172A patent/CA2144172A1/en not_active Abandoned
  • 1993-09-10 JP JP6506692A patent/JPH08501536A/ja active Pending
  • 1993-09-10 IL IL10698093A patent/IL106980A/xx not_active IP Right Cessation
  • 1993-09-10 CN CN93119099A patent/CN1088046A/zh active Pending
  • 1993-09-10 EP EP93920592A patent/EP0668718A4/en not_active Withdrawn
  • 1993-09-10 WO PCT/AU1993/000465 patent/WO1994005150A1/en not_active Ceased
  • 1993-09-27 TW TW082107955A patent/TW230186B/zh active

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