CN1624101A - Naegleria sp. and its application - Google Patents

Abstract

A Naegleria sp. W2 (CGMCC No.1044) with strong algophagous power is disclosed. It can be used for treating the nutrients-enriched water in river or lake as it can eat multiple kinds of cateniform blue algae.

Description

A kind of Na Shi worm and application thereof

Technical field

The present invention relates to a kind of protozoon and application thereof, particularly a kind of Na Shi worm and application thereof.

Background technology

Lake eutrophication problem serious day by day often causes the blue-green alga bloom outburst.Blue-green alga bloom is a typical ecological problem, and the key of control and improvement algal tufa is to find a kind of factor that can directly effectively kill or suppress algal grown, and this factor should be economical, is convenient to extensive use, and can not causes secondary pollution.Wawter bloom can be effectively controlled in the use of chemistry algicide in one period, but costs an arm and a leg, and may produce secondary pollution.Even chemical algicide does not produce negative impact at once, also can increase the concentration of objectionable impurities in the bottom mud in lake.Separate the biology control algae factor (mainly being microorganism) obtain and not only can not produce the problems referred to above from water body, and their specificitys acting on target organisms, is a kind of control algae factor that gets a good chance of.

Occurring in nature has a lot of biologies can control algae grows, and they mainly comprise: phycovirus (phycophage), molten algae bacterium, protozoon, fungi and actinomycetes (progress China virusology 1,999 14 (2): the 100-104 of phycovirus (phycophage) such as Zhao Yijun stone is just beautiful; The research trends hydrobiology of the basis of Liu Zhao Yijun Yongding harmful algae and microbial control thereof-phycomycete relation).They directly are located away from nature, therefore use these biological control algae factors just original lake ecological equilibrated to be changed, can not resemble nutrition source control (descending regulation and control) and " bio operon " and (Biomanipualtion) lake ecosystem be produced basic change.They are adapted at the wawter bloom early period of origination and use, and control the algae bio amount in a short time or stop algae to breed in a large number.

The environment-adapting ability of more various control algae biotic factors, fecundity, control algae effectiveness, host range, the adaptive faculty that the host is changed and the multiple factors such as resistance under the adverse environment, it is believed that protozoon is a kind of control algae agent (Daft that practical potentiality are arranged very much, M.J., Burnham, J.C., and Yamamoto, Y.1985.Algalblooms:consequences and potential cures.J.Appl.Bacteriol. (Symp.Suppl.): 175-186).Protozoon has following advantage as the control algae factor: it is wide, heavy that protozoon is got the food scope, observes in the laboratory, as long as the protozoon number reaches a certain threshold value, the frustule in the system can rapid approach exhaustion; A lot of protozoons can form packing when food exhausts, when algae was increased again, packing can become food algae nourishing body by broken wall again; The packing structure has very strong resistance, and this form is packing and transportation easily; Breeding easily, protozoon can utilize organic substratum bulk fermentation to cultivate.

Occurring in nature, it can be food with the algae that the protozoon of many types is arranged, even some protozoons is arranged with the unique food source of algae as them.With regard to present report, protozoon mainly is divided into the food algae: flagellate, amoeba and three big classes of ciliate.

Naegleria in two flagellum Amoebidaes (Naegleria Sp.) amoeba, multiply in fresh water more, the active trophont is long amoeba shape, size is 7 * 20 μ m, the Chang Xiangyi end stretches out roomy bold and unrestrained pseudopodium, the other end is more tiny to be pseudo-tail region, can be formed with the trophont of 2 flagellums in poor environment, and this kind of does not divide and does not directly form packing yet.The packing circle, diameter 9 μ m, monokaryon, cyst wall is smooth porose.

Blue algae is widely distributed, mainly lives in the fresh water, can form ' wawter bloom ', and the wawter bloom that is formed by blue algae can be seen throughout the year in China south.Wherein, anabena or plan anabena wawter bloom are caused by the kind and the plan anabena of spiral anabena (Anabaenaspiroides) or other Anabaena.Sociales are very outstanding, can account for more than 95% of biological total amount, and water colour is bluish-green or dark green, can see emerald green wadding yarn or blue-greenish colour epistasis.

The innovation and creation content

The purpose of this invention is to provide a kind of new Na Shi worm that eats the algae ability more by force that has.

Na Shi worm provided by the present invention is Na Shi worm (Naegleria sp.) W2, is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on November 25th, 2003, and preserving number is CGMCC № 1044.

Na Shi worm (Naegleria sp.) W2 CGMCC № 1044 obtains from the separation of Dian Chi wawter bloom water sample.

Experimental results show that, Na Shi worm (Naegleria sp.) W2 CGMCC № 1044 can get the multiple chain blue-green algae of food, can use separately, as with it attached on the floating matter (such as grass stalk or barley), they itself promptly have the effect of control algae, produce effect preferably, also can use jointly simultaneously with control algae factors such as other protozoon, molten phycomycete and phycophages, can produce and more fully control the algae effect, effectively administering body eutrophication, particularly the blue-green alga bloom aspect will play a significant role, and market potential is huge.

Description of drawings

Fig. 1 a is that Na Shi worm W2 goes up food algae spot and the blank that forms at wawter bloom anabena (Anabaena flos-aquae)

Fig. 1 b is Na Shi worm W2 goes up the food algae spot that forms at wawter bloom anabena (Anabaena flos-aquae) the changing conditions of being separated by 6 hours

The molten algae situation that Fig. 2 causes in the anabena nutrient solution for Na Shi worm W2

Fig. 3 A is the nourishing body of Na Shi worm W2 under the differential interference visual field

Fig. 3 B is the packing of Na Shi worm W2 under the differential interference visual field

Fig. 4 is the get food process of same protozoon nourishing body under the differential interference visual field to an anabena cytofilament

Fig. 5 gets the Photomicrograph of food Na Shi in period worm W2 under common bright field (BR) and fluorescence dark field (FL) for difference

Fig. 6 is the pcr amplification of protozoon genomic dna and 18SrRNA gene thereof

Fig. 7 is the comparison of Na Shi worm W2 and Naegleria australiensis 18SrRNA gene

Fig. 8 carries out 60 hours photo for Na Shi worm W2 food algae process experiment in the triangular flask

Fig. 9 a is that Na Shi worm W2 eats chlorophyll-a change curve (representing the frustule biomass) in the algae process experiment in the triangular flask

Fig. 9 b is that Na Shi worm W2 eats protozoon number logarithmic curve in the algae process experiment in the triangular flask

Fig. 9 c is that Na Shi worm W2 eats pH value change curve in the algae process experiment in the triangular flask

Fig. 9 d is that Na Shi worm W2 eats dissolved oxygen (DO) change curve in the algae process experiment in the triangular flask

What Figure 10 was Na Shi worm W2 to the anabena suspension culture removes the algae effect

Figure 11 is the variation of Chl-a content in the Na Shi worm W2 food algae process

Embodiment

The separation of embodiment 1, protozoon Na Shi worm (Naegleria sp.) W2 CGMCC № 1044 and food algae characteristic

1. the separation of Na Shi worm (Naegleria sp.) W2 CGMCC № 1044

Dian Chi wawter bloom water sample through after host algae-wawter bloom anabena (Anabaena flos-aquae) cell cultures liquid enrichment, is separated with algae tongue dilution coating method.After the dull and stereotyped cultivation of algae tongue 4-5 days, form the food algae spot that constantly enlarges on the algae tongue, shown in Fig. 1 a and Fig. 1 b, among Fig. 1 a, the food algae spot that the left side culture plate forms on the algae tongue after 4 days for cultivation; The right side culture plate is a blank; Among Fig. 1 b, the left side culture plate is the food algae spot before 6 hours, and the right side culture plate is the food algae spot after 6 hours.Through microscopical determination, what form food algae spot is a kind of amoeba, i.e. Na Shi worm (Naegleria sp.) W2 CGMCC № 1044.To eat the algae spot and choose to receive in the algae liquid with toothpick and can cause molten algae, as shown in Figure 2, among Fig. 2, the right side culturing bottle is for inserting 96 hours molten algae situations of 0.5ml Na Shi worm (Naegleria sp.) W2 CGMCC № 1044 cultures; Middle culturing bottle is for inserting 48 hours molten algae situation of 0.5ml W2 culture; The left side culturing bottle is a blank.

2. the food algae scope of Na Shi worm (Naegleria sp.) W2 CGMCC № 1044

The different algal species culture is made algae tongue flat board, on the algae tongue, form its food algae scope of capability evaluation of food algae spot according to Na Shi worm (Naegleria sp.) W2 CGMCC № 1044.The result is as shown in table 1:

Table 1. Na Shi worm (Naegleria sp.) W2 CGMCC № 1044 and other two kinds are primary

Animal is to the food situation of the getting * of various blue-green algaes

????Cyanobacteria	Food algae spot diameter *
					????W2	????W3	????D5
????Anabaena?cylindrical	????+	????++	????++
				????Anabaena?flos-aquae	????++	????++	????+
????Aphanizomenon?flos-aquae	????-	????++	????-
				????Aphanocapsa?elachista	????++	????+	????+

????Cylindrospermum?majus	????+	????-	????-
				????Gloeotrichia?echinulata	????++	????++	????++
????Phormidium?foveolarum	????+	????++	????++
				????Anacystis?nidulans	????-	????-	????-
????Oscilltoria?tenuis	????-	????-	????-
				????Microcystis?aeruginosa	????-	????-	????-

Annotate: * cultivates the size of record food algae spot after 6 days :-, do not form food algae spot; +, food algae spot diameter≤15mm; ++, food algae spot diameter＞15mm.

Table 1 shows that Na Shi worm (Naegleria sp.) W2 CGMCC № 1044 can get the multiple chain blue-green algae of food, and single celled Anacystis nidulans Anacystis nidulans and Microcystis aeruginosa Microcystis aeruginosa are not had feeding effect.In the table 1, W3 and D5 are isolating two kinds of food algae protozoons from the Beijing area, belonging to Filosea (Filosea), to suck the protozoon animal they and the algae that bite this genus of section (Vampyrelldae) nuclear Eimeria (Nuclearia) in close relations, often with algae symbiosis or parasitic and algae on, be main food with algae.

3. the morphological specificity of Na Shi worm (Naegleria sp.) W2 CGMCC № 1044

By microscopic examination, determined the morphological specificity of Na Shi worm (Naegleria sp.) W2 CGMCC № 1044, as Fig. 3 A, Fig. 3 B and shown in Figure 4, ellipse during protozoon Na Shi worm (Naegleria sp.) W2 CGMCC № 1044 rests, diameter 10-30um, can be in motion or when predation with the transparent pseudopodium that produces indefinite form, this moment health diameter 20-50um.Monokaryon (diameter 4-6um) is got the food back and is produced food vacuole (3-10um), and food exhausts back nutrition cognition and forms packing (diameter 10-20um).Among Fig. 3 A, N is a nucleus, and V is a food vacuole, and P is a pseudopodium; Among Fig. 3 B, C is a packing; CW is a cyst wall; Scale is represented 10um among Fig. 3 A and Fig. 3 B.Among Fig. 4, picture A is that a protozoon moves towards one section along algal filament; Picture B is that Na Shi worm (Naegleria sp.) W2 CGMCC № 1044 pseudopodium extend an end that has surrounded algal filament; Picture C and D are that algal filament is eaten by protozoon; V is a food vacuole; P is a pseudopodium; F is the anabena cytofilament, and scale is represented 10um among the figure.

Excite down at 430nm wavelength light, phycoerythrobilin in the frustule and chlorophyll can be inspired red fluorescence, according to this principle, followed the tracks of frustule in the intravital mobile and digestion situation of protozoon, the result as shown in Figure 5, expression frustule digestion process in the food vacuole in Na Shi worm (Naegleria sp.) W2 CGMCC № 1044 bodies: inserted W2 as can be seen 24 hours from picture A and B, there are a large amount of algal filaments, protozoon quantity does not also get on, protozoon food is abundant, and whole cell paste all sends the red fluorescence of frustule; Insert Na Shi worm (Naegleria sp.) W2 CGMCC № 1,044 48 hours as can be seen from picture C and D, protozoon quantity significantly improves, and contains in the cell paste to send the bright food vacuole of red fluorescence in a large number; Insert Na Shi worm (Naegleria sp.) W2 CGMCC № 1,044 96 hours as can be seen from picture E and F, the food in the system only remains a large amount of algae bones of the body (CD) near exhausting, and a lot of nourishing bodys have not had food vacuole.Among Fig. 5, F is an algal filament; V is a food vacuole; P is a pseudopodium; N is a nucleus; T is Na Shi worm (Naegleria sp.) W2 CGMCC № 1044 nourishing bodys, and mark is represented 20um among the figure.

4. determine the classification position of W2 by rDNA gene identification technology

Protozoic in the past microscopic pattern structure and life characteristic are the main standard of its evaluation, but will determine that affiliated kind needs rich experience and long time by morphological structure for the protozoon that a kind of new separation obtains.The conservative gene of coding rRNA in the biomass cells has become the important sign of determining the species classification position and the important parameter of studying molecular evolution.Because molecule clone technology is increasingly mature, it is very simple and efficient to obtain protozoic rDNA sequence.

With food algae protozoon genomic dna is template, is that primer carries out pcr amplification with W2-A (5 '-TGTTGAGTCAA ATTAAGCCGC-3 ') and W2-S (5 '-AATTAGGGTTCGATTCCGGA-3 ').Amplified production separates through 1.0% agarose gel electrophoresis, and the result shows that each food algae protozoon genomic dna all increases out and specific bright band occurred that its size is all consistent with the 930bp that establishes expectation as shown in Figure 6.M represents λ DNA/EcoR I+HindIII Marker among Fig. 6; The A road is the result of anabena contrast pcr amplification; Na Shi worm (Naegleria sp.) W2 CGMCC № 1044, W3,2H, D5 (W3,2H, D5 are three kinds of food algae protozoons, belong to Filosea (Filosea) suck bite section (Vampyrelldae) nuclear Eimeria (Nuclearia)) be four kinds of protozoic genomic dnas of food algae; The 1-8 road is for to do the 18SrRNA gene PCR product of template with the genomic dna of Na Shi worm (Naegleria sp.) W2 CGMCC № 1044, W3,2H, D5 respectively.

Cloning and sequencing result to various food algae protozoon 18SrRNA gene PCR products carries out the BLAST analysis, the result shows that the similarity of the 18SrRNA Gene Partial sequence of Na Shi worm (Naegleria sp.) W2 CGMCC № 1044 18SrRNA Gene Partial sequences and Australian Na Shi worm (Naegleria australiensis) reaches 99% as shown in Figure 7.In conjunction with microscopic examination and macroscopic view experiment, the form of W2 and the life history characteristic all meet the feature of Naegleria, determining that Na Shi worm (Naegleria sp.) W2 CGMCC № 1044 belongs to splits Gorgon euryale order (Schizopyrenida) Naegleria (Negleria).

Na Shi worm (Naegleria sp.) is also referred to as Fu Shi Nei Geli amoeba, is the very wide class protozoon that distributes in soil and the water surrounding.Research to the Na Shi worm at present is primarily aimed at the pathogenic of it, Fu Leer-Nai Geli (Naegleria fowleri) protozoon can cause that primary amebic meningoencephalitis (primary amebicmebubgo-encepha-litis) and W2 CGMCC № 1044 18SrRNA Gene Partial sequence similarities reach 99% Australian Na Shi worm (Naegleria australiensis) and also can cause the suppurative amebic meningitis of people or other animals.The W2 CGMCC № 1044 that studies in the laboratory does not find any pathogenic of tool so far yet, and abroad this is belonged in the protozoic research also never relevant for the active report of its food algae.This genus protozoon mainly is divided into 24 kinds according to rDNA (5.8S DNA or 18SrDNA) sequence ^[7]Wherein with the rDNA sequence similarity of W2 the highest be 99% (difference that 4 bases are arranged in the sequence of 923 bases), W2 CGMCC № 1044 is 97% (difference that 20 bases are arranged in the sequence of 923 bases) with the similarity of generic Naegleria.gruberi 18S subunit ribosomal RNA gene, with the similarity of generic Naegleria italicasubunit ribosomal RNA gene be 97% (difference that 23 bases are arranged in the sequence of 923 bases), with the similarity of Naegleria clarki be 96% (difference that 24 bases are arranged in the sequence of 717 bases), W2 CGMCC № 1044 and Naegleria fowleri, Naegleria indonesiensis, Naegleria fowleri, Naegleria lovaniensis, Naegleria andersoni and Naegleria jamiesoni etc. belong to the rDNA sequence similarity of kind together between 96%-94%.For very conservative ribosome-RNA(rRNA) encoding gene, such difference has been enough to become the evidence that W2 is a novel species.

The experiment of embodiment 2, W2 simulation control algae

(1) Na Shi worm (Naegleria sp.) W2 CGMCC № 1044 food algae processes in the triangular flask

Experiment is carried out in the 300ml triangular flask, every bottle of 120ml anabena algae liquid.To initial chlorophyll A content is 0.95mg l ^-1Anabena algae liquid in add Na Shi worm (Naegleria sp.) W2 CGMCC № 1044 nutrient solutions, make Na Shi worm (Naegleria sp.) W2 CGMCC № 1044 initial concentrations be respectively 100,500,1000 cell/ml.Monitor anabena biomass, active protozoon quantity, dissolved oxygen and pH value every day.Experimental result shows in 3 day time that shown in Fig. 8, Fig. 9 a, Fig. 9 b, Fig. 9 c and Fig. 9 d Na Shi worm (Naegleria sp.) W2 CGMCC № 1044 breeds in a large number, when reaching 10 ⁵During individual cell/ml, the frustule biomass sharply reduces, and the dissolved oxygen and the pH value of system all descend simultaneously.After 4 days, as the frustule of Na Shi worm (Naegleria sp.) W2 CGMCC № 1044 foods seldom, most of Na Shi worm (Naegleria sp.) W2 CGMCC № 1044 nourishing bodys begin to form packing, enter dormant state, and dissolved oxygen gos up to some extent owing to the reoxygenation effect in the system.Among Fig. 8, three bottles of protozoon starting point concentrations of A row are 2000 cell/ml; Three bottles of protozoon starting point concentrations of B row are 500 cell/ml; Three bottles of protozoon starting point concentrations of C row are 100 cell/ml; The D row are blank for three bottles.Each point is represented three groups of multiple mean values among Fig. 9 a, Fig. 9 b, Fig. 9 c and Fig. 9 d, and variance marks with error line.

(2) W2 removes the algae effect to anabena simulation wawter bloom

Be sub-packed in the tank each 2 liters behind 3 days the anabena culture mixing with cultivating, add 0,0.004,0.04,0.4 respectively, (2-3/ul), measured the variation of molten algae efficient and chlorophyll-a (Chl-a) content thereof every 12 hours of Na Shi worm (Naegleria sp.) W2 CGMCC № 1044 suspension of 4.0ml.The result as shown in Figure 10 and Figure 11, Figure 10 shows that the protozoon number reaches 10 in the system ⁵After the cells/ml, the frustule biomass can sharply descend (handled from 48 hours and the difference of processing in 96 hours as can be seen), reaches except that the algae effect.Figure 11 shows that along with the increase of Na Shi worm (Naegleria sp.) W2 CGMCC № 1044 concentration, the content of chlorophyll-a constantly descends.

Claims (3)

1, Na Shi worm (Naegleria sp.) W2 CGMCC № 1044 and mutant and varient.

2, the application of Na Shi worm (Naegleria sp.) W2 CGMCC № 1044 in administering body eutrophication.

3, the application of Na Shi worm (Naegleria sp.) W2 CGMCC № 1044 in administering blue-green alga bloom.

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