CN100432211C

CN100432211C - Standardization generation and proliferation method by stimulating cryptonucleus insect and its special collector

Info

Publication number: CN100432211C
Application number: CNB2006100355752A
Authority: CN
Inventors: 李安兴; 但学明
Original assignee: National Sun Yat Sen University
Current assignee: Sun Yat Sen University; National Sun Yat Sen University
Priority date: 2006-05-23
Filing date: 2006-05-23
Publication date: 2008-11-12
Anticipated expiration: 2026-05-23
Also published as: CN1912099A

Abstract

The invention relates to Cryptocaryon irritans standardization passage fertilizing method and its special collector which belongs to aquatic animal field. The method includes the following steps: using Trachinotus ovatus as host; infecting as dosage for 8000-15000 larva per fish at 27+/-0.5 degree centigrade water temperature 29-32 per mill salinity between 22:00-24:00 for 3h in dark; appearing trophont after 60h which drops to form cyst prosoma in 24h; collecting and culturing for 60h; hatching larva reaches climax after 24h; collecting the larva. And it has the advantages of high overall yield for parasite, little used times, and simple collecting method.

Description

A kind of stdn passage fertilizing method and special collector thereof that stimulates cryptonucleus insect

Technical field

The present invention relates to the hydrocoles field, be specifically related to a kind of stdn passage fertilizing method and special collector thereof that stimulates cryptonucleus insect.

Background technology

Stimulating cryptonucleus insect (Cryptocaryon irritans Brown 1951) is a kind of ciliate that colonizes on the torrid zone, the subtropics seawater fish body, causes " ichthyophthiriasis " of seawater fish.In recent years, stimulate cryptocaryoniosis, endanger seriously day by day, still up to the present, also do not find effective means thoroughly to prevent and treat this parasitosis in frequently outburst all over the world.It is very difficult will obtaining enough materials at any time in the research that stimulates cryptonucleus insect, is to be subjected to strict restriction in season because stimulate the outburst of cryptocaryoniosis under the natural condition, and the uncertainty of illness outbreak causes the acquisition of material not ensure; And this Eimeria is in the fish obligatory parasite, the life history essential principal host fish participation, can not carry out vitro culture, so Many researchers once attempted to set up this parasitic system that goes down to posterity of cover in the laboratory.Colorni 1985 is the propagation that the host has carried out this worm with golden porgy (Sparus aurata) in the laboratory first, but infects not quantitatively, and productive rate is also unclear; Burgess etc. 1994 with grey mullet (Chelon labrosus) as the host, in the laboratory, successfully set up the system that goes down to posterity of 7 strain systems of this worm, but the propagation of each worm strain is all failed to surpass 24 and is taken turns circulation, the parasite productive rate is also very low, maximum has only 11.7 times, and the low-level infection rate of larva is the major cause that causes poor efficiency; Yoshinaga etc. 1994 have successfully carried out the propagation of this worm with black Medaka (Poecilia latipinna) as the host, but a little less than the parasite reproductivity, every fish can only be received 50～100 trophonts.Simultaneously, these authors do not carry out stdn to collection, infection, the breeding of polypide.These defectives have all limited parasitic ultimate capacity, and the parasite quantity that makes acquisition has caused severely restricts to research work very little.

Summary of the invention

The objective of the invention is to overcome existing the stimulation in the cryptonucleus insect research and have the insufficient problem of material, a kind of standardized method that stimulates cryptonucleus insect to go down to posterity and breed is provided, study so that obtain enough stimulation cryptonucleus insect materials at any time.

Another object of the present invention provides the special collector that is used for above-mentioned stimulation cryptonucleus insect passage fertilizing method.

To achieve these goals, the used host fish of the stdn passage fertilizing method of stimulation cryptonucleus insect of the present invention adopts Trachinotus ovatus (Trachinotus ovatus).Under 27 ± 0.5 ℃ of water temperatures, salinity 29～32 ‰ conditions, at night between 22:00～24:00, infected the fish body in the dark 3 hours with taking off the dosage of the larva of bag in 2 hours by every fish 8000～15000 larvas, obvious visible trophont appearred on the fish-skin skin and the gill after 60 hours, nearly all trophont all comes off from the fish body and forms the packing precursor in ensuing 24 hours, collect the packing precursor that comes off with special collector, cultivate after 60 hours, packing begins to discharge larva, the larva hatching peaked after 24 hours, collected larva this moment.Finish one and take turns above-mentioned circulation 1 week just, one takes turns circulation can receive about 1,000 ten thousand larvas (each host fish of taking turns that circulation infects is 10 tails), and productive rate is up to 122 times.

Above-mentioned larva of collecting can be carried out next round circulation, each group fish can consecutive infection 4 times, and the 1st subinfection dosage is 8000 larvas. fish ^-1, the 2nd subinfection dosage is 10000 larvas. and fish ^-1, the 3rd subinfection dosage is 12,000 larvas. and fish ^-1, the 4th infective dose is 15000 larvas. and fish ^-1Because the metainfective parasite productive rate of the 4th significantly descends, therefore infect next time and change one group of fresh fish.

Above-mentioned special collector comprises bucket, funnel, beaker, and funnel places bucket inner, and funnel bottom is beaker fixedly.Also be provided with water inlet pipe, rising pipe and aerogenesis apparatus in the bucket.Water inlet pipe and rising pipe can maintain miniflow water, and aerogenesis apparatus can keep inflation, helps the existence of fish.

Compared with prior art, the present invention has following beneficial effect: at first, owing to selected for use Trachinotus ovatus as the outstanding host who stimulates cryptonucleus insect, parasitic productive rate reaches as high as 122 times, minimumly also can reach 60.7 times, therefore parasitic ultimate production is very high, and each circulation can both be received about 1,000 ten thousand larva from 10 tail host fish bodies.Secondly, this systemic circulation cycle is short, under 27 ± 0.5 ℃, and every just colored week age of circulation of taking turns.Once more, the material collection method is simple, and dead larva can kill the back by centrifugal collection with formalin, and the larva that lives can be collected with membrane filtration, and particularly a large amount of packing precursors can be collected easily with special collector.

Description of drawings

Fig. 1: Trachinotus ovatus is stimulated the larva of cryptonucleus insect to infect after 60 hours, and tangible white trophont (photo) has appearred in body surface;

Fig. 2: Trachinotus ovatus is stimulated the larva of cryptonucleus insect to infect after 60 hours, has occurred tangible white trophont (photo) on the fish gill;

Fig. 3: the special collector synoptic diagram of packing precursor;

Fig. 4: a large amount of packings that use special collector to collect tightly are attached to beaker bottom (photo).

Wherein, among Fig. 3,1 barrel, 2 funnels, 3 beakers, 4 water inlet pipes, 5 rising pipes, 6 aerogenesis apparatus.

Embodiment

1, go down to posterity and use fish: the host fish that uses in going down to posterity is Trachinotus ovatus (Trachinotusovatus), counterpoise 167.8 ± 15.1g.

2, the processing of seawater: seawater adopts natural sea-water, salinity 29～32 ‰, and dissolved oxygen=5.0mg/L, pH 7.9～8.3, nitrite＜0.1mg N/L.Cultivate parasitic seawater and filter earlier, the 30min that sterilizes in 120 ℃ again for preventing infectation of bacteria, adds 100IU.ml before using ^-1Penicillin and 100 μ l.ml ^-1Streptomycin sulphate.

3, the go down to posterity stdn of temperature and salinity: 27 ± 0.5 ℃ of the water temperatures that goes down to posterity, sea water salinity 29～32 ‰.

4, the stdn of infection time and infective dose: infect Trachinotus ovatus with taking off the cryptocaryon irritans larva that wraps in 2 hours, infect at night and carry out during 22:00～4:00, the water yield is controlled at the 5L. fish during infection ^-1, fish infects after 3 hours in the dark puts back to big aquifer cultivation.As shown in Figure 1, 2, the Trachinotus ovatus body surface and the fish gill that is infected by cryptocaryon irritans larva tangible white trophont occurred after infecting 60 hours.

5, the collection method of the making of special collector and packing precursor: larva infection fish grows after 60 hours and is sophisticated trophont, and breaking away from the fish body then becomes the packing precursor, and we collect the packing precursor that comes off with special collector.As shown in Figure 3, place a suitable funnel 2 that is made by slick plastic sheet for 1 li at the plastic tank of 160L, the beaker 3 of a 2L is fixed in funnel 2 bottoms, is provided with water inlet pipe 3, rising pipe 4 in the bucket 1 and can produces the gas stone 5 of gas.Treat on the fish gill and the body surface to occur (seeing Fig. 1,2) behind the tangible white trophont, fill it up with seawater for 2 li, again fish is put into funnel, keep inflation and miniflow water toward plastic tank 1 and the funnel of 160L.For preventing the parasite escape, the water surface will be lower than the upper limb of funnel 2, and the packing precursor that comes off sinks to beaker 3 bottoms, sticks to beaker 3 bottoms and forms packing, just can hatch for 3 li at beaker after the purged of impurities.Because parasite is many, so hatching the time will be inflated, and every day, 8:00 changed the seawater of once sterilizing.

6, the collection method of larva: the packing precursor that special collector is received begins to hatch larva after through 2 days cultivation, the bag that takes off of larva peaks after 1 day, collect worm liquid this moment, draw 5 parts (every part 50 μ l) behind the mixing, add 10 μ l formalin and kill larva, at the microscopically counting, compute goes out the concentration of larva.These larvas can be used for carrying out next round and go down to posterity, and unnecessary larva can collect and be used for other experiment, if collect dead larva, then use 100 μ l.L ^-1Formalin larva is killed, with whizzer (KUBOTA 5100) 3500rpm centrifugal 10min, be collected in the 1.5ml centrifuge tube-80 ℃ of preservations.If collect the larva that lives, be the membrane filtration worm liquid of 0.45 μ l directly then with the aperture, larva concentration can reach 200,000 larva .ml ^-1

Claims

1, a kind of passage fertilizing method that stimulates cryptonucleus insect, comprise cryptocaryon irritans larva infection host fish, collect the packing precursor, the hatching larva, it is characterized in that used host fish is a Trachinotus ovatus, 26.5～27.5 ℃ of water temperatures, under sea water salinity 29～32 ‰ conditions, at night between 22:00～24:00, infect the fish body in the dark with taking off the dosage of the larva of bag in 2 hours by 8000～15000 larvas of every fish, when treating on the fish gill and the body surface to occur obvious visible trophont, collect the packing precursor that comes off and form from the fish body with special collector, hatching packing precursor is collected the larva that the packing precursor discharges.

2, the passage fertilizing method of stimulation cryptonucleus insect as claimed in claim 1 is characterized in that and the larva of collecting can be infected Trachinotus ovatus again that propagation goes down to posterity next time.