CN108037121B - Method for accurately measuring real pH value of insect intestinal environment - Google Patents
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- CN108037121B CN108037121B CN201711116143.9A CN201711116143A CN108037121B CN 108037121 B CN108037121 B CN 108037121B CN 201711116143 A CN201711116143 A CN 201711116143A CN 108037121 B CN108037121 B CN 108037121B
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- 241000238631 Hexapoda Species 0.000 title claims abstract description 72
- 230000000968 intestinal effect Effects 0.000 title claims abstract description 43
- 238000000034 method Methods 0.000 title claims abstract description 18
- 210000001035 gastrointestinal tract Anatomy 0.000 claims abstract description 50
- 238000012360 testing method Methods 0.000 claims abstract description 11
- 239000006228 supernatant Substances 0.000 claims abstract description 7
- 241000659518 Lozotaenia capensana Species 0.000 claims description 9
- 210000000936 intestine Anatomy 0.000 claims description 5
- 210000003238 esophagus Anatomy 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 238000007598 dipping method Methods 0.000 claims description 2
- 102000004190 Enzymes Human genes 0.000 abstract description 16
- 108090000790 Enzymes Proteins 0.000 abstract description 16
- 244000005700 microbiome Species 0.000 abstract description 11
- 235000013305 food Nutrition 0.000 abstract description 6
- 238000011160 research Methods 0.000 abstract description 6
- 238000011161 development Methods 0.000 abstract description 3
- 230000029087 digestion Effects 0.000 abstract description 3
- 235000016709 nutrition Nutrition 0.000 abstract description 3
- 230000035764 nutrition Effects 0.000 abstract description 3
- 239000000575 pesticide Substances 0.000 abstract description 3
- 229940088598 enzyme Drugs 0.000 description 15
- 230000000694 effects Effects 0.000 description 14
- 108091005804 Peptidases Proteins 0.000 description 6
- 239000004365 Protease Substances 0.000 description 6
- 244000144730 Amygdalus persica Species 0.000 description 4
- 102000035195 Peptidases Human genes 0.000 description 4
- 235000006040 Prunus persica var persica Nutrition 0.000 description 4
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 3
- 230000002503 metabolic effect Effects 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 235000019419 proteases Nutrition 0.000 description 3
- 229910021642 ultra pure water Inorganic materials 0.000 description 3
- 239000012498 ultrapure water Substances 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 2
- 108091007734 digestive enzymes Proteins 0.000 description 2
- 102000038379 digestive enzymes Human genes 0.000 description 2
- GPRLSGONYQIRFK-UHFFFAOYSA-N hydron Chemical compound [H+] GPRLSGONYQIRFK-UHFFFAOYSA-N 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 210000003750 lower gastrointestinal tract Anatomy 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 210000002438 upper gastrointestinal tract Anatomy 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 108010065511 Amylases Proteins 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102000004308 Carboxylic Ester Hydrolases Human genes 0.000 description 1
- 108090000863 Carboxylic Ester Hydrolases Proteins 0.000 description 1
- 102000005575 Cellulases Human genes 0.000 description 1
- 108010084185 Cellulases Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000659001 Grapholitha molesta Species 0.000 description 1
- 241001147381 Helicoverpa armigera Species 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 241000254023 Locusta Species 0.000 description 1
- 102100029677 Trehalase Human genes 0.000 description 1
- 108010087472 Trehalase Proteins 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 229940025131 amylases Drugs 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 235000021258 carbohydrate absorption Nutrition 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 108091007735 digestive proteases Proteins 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000010794 food waste Substances 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- -1 hydrogen ions Chemical class 0.000 description 1
- 210000003405 ileum Anatomy 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
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- 235000019421 lipase Nutrition 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
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- 235000019833 protease Nutrition 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 210000000664 rectum Anatomy 0.000 description 1
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- 235000013343 vitamin Nutrition 0.000 description 1
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- 229930003231 vitamin Natural products 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
- G01N21/80—Indicating pH value
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- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for accurately measuring the real pH value of an insect intestinal environment, which comprises the steps of firstly obtaining the insect intestinal tract, collecting a certain amount of insect intestinal tract and the content thereof, grinding the insect intestinal tract and the content thereof by using a biological tissue grinder, centrifuging by using a cold centrifuge to obtain the supernatant of the insect intestinal tract and the content thereof, and quickly measuring the pH value of the supernatant by using wide pH test paper and precise pH test paper, so that the real pH value of the insect intestinal environment can be obtained, the real pH value of the insect larva intestinal environment can be accurately obtained in a short time, convenience is provided for the subsequent research of insect intestinal microorganisms and intestinal enzyme systems, and the method has the characteristics of simple operation and real and reliable measured values. Has important guiding value for the food digestion and nutrition acquisition of insects and the development of biological pesticides related to insect midgut.
Description
Technical Field
The invention belongs to the technology of insect intestinal environment determination, and particularly relates to a method for accurately measuring the real pH value of an insect intestinal environment.
Background
The digestive tract of insects has the functions of digesting food particles, absorbing nutrients, maintaining ion balance in the body and discharging metabolic waste. It is divided into foregut, midgut and hindgut according to its function. Among them, the foregut has a crop for the temporary storage of food, and the midgut is the site where intestinal commensals and digestive enzymes and proteases mainly exist, with the most prominent function. The middle intestine primarily secretes digestive juices, digests food, and absorbs nutrients. The main function of the hindgut (including the ileum and rectum) is to exclude nitrogenous metabolic wastes and food residues, while providing a favorable nutrient environment for the insect gut flora.
The insect intestinal tract is the most important component of the insect digestive system, and is a dynamic and stable environment accompanied with various vital metabolic activities of insects, and the complex intestinal environment of insects provides a superior colonization environment for certain microorganisms. A large number of intestinal microorganisms exist in the intestinal environment, and partial researches show that the existence of the microorganisms has great significance on normal life activities of host insects. Most microorganisms are in a mutualistic symbiotic relationship with host insects, and they contain multiple enzyme systems that also play important roles in vitamin synthesis, fat and carbohydrate absorption and utilization. The intestinal microorganisms and the insect intestinal tract influence each other to finally reach dynamic equilibrium, so that the insect intestinal environment is maintained at a constant pH value.
Because insects have diversified ecological habits and different feeding characteristics, intestinal flora of different types of insects also has diversified characteristics. The intestinal environment is greatly different due to different insect species, which is also a long-term co-evolution result of insects adapting to various different ecological niches and feeding habits, and the co-evolution gradually evolves to a phenomenon that specific intestinal parts of insects colonize and fix intestinal microorganisms. Therefore, different species of insect intestinal tracts colonize different intestinal microorganisms, the species of the intestinal microorganisms can affect the actual pH value of the intestinal environment, and the actual pH value of the intestinal environment can be affected to a certain extent by eating different host plants. The stable pH value of the intestinal environment has an important function in the aspect of maintaining normal physiological activities of insects, can resist invasion and colonization of foreign bacteria to a certain extent, and plays an important role in promoting the function of an immune system.
Most proteases and peptidases in insects are secreted directly into the midgut, and some of the enzyme systems in insects remain in the midgut cells or in association with the outer membrane, so that the pH in the intestinal lumen directly affects the level of enzyme activity, to an extent dependent on the pH optimum for enzyme activity. And the difference of intestinal pH environment among different insects is large. The method has practical application value in researching the intestinal enzyme activity of the insects under the condition of the real pH value of the intestinal tracts of the insects, and can reflect the real enzyme activity change. The variety of enzymes in the insect intestinal environment is large, and in recent years, digestive enzymes, proteases, amylases, cellulases, lipases, carboxylesterases, trehalases, various midgut receptor proteins, and the like have been most studied. The functions of the series of enzymes play a remarkable role in maintaining normal metabolism and physiological activities of insects, and the real pH value of the intestinal environment of the insects has a direct influence on the activity of the enzymes. The series of researches on the enzyme activity related to the insect intestinal tract have to be established on the basis of the real pH value of the insect intestinal tract environment so as to be more practical and valuable.
In recent years, research on a series of enzyme activities in insect intestinal microorganisms and insects has become a new focus of general attention of researchers. Therefore, the method for rapidly measuring the real and accurate pH value of the insect intestinal tract is particularly important in the aspect of physiological and biochemical research of insects, and has important guiding values for food digestion and nutrition acquisition of insects and development of biological pesticides related to the insect midgut.
The conventional methods related to the measurement of the pH value of the intestinal tract of the insect mainly comprise the following two types, namely the method of the yellow gentamian and the like (2009) in the document of 'influence of pH on the activity of the major protease of the intestinal tract of a locusta pratensefolia' is as follows: "the midgut portion was immediately trimmed and the midgut and its contents were eluted in a small beaker with 2mL of ultrapure water, and the pH of the midgut intestinal fluid was immediately measured at room temperature with a pH meter. "pH is a hydrogen ion concentration index (hydrogen ion concentration) refers to the ratio of the total number of hydrogen ions in a solution to the amount of total substances. In the method, the pH value of the middle intestine and the content thereof is eluted in a beaker by 2mL of ultrapure water, and the original H ion concentration in the intestinal tract solution is diluted due to the addition of the ultrapure water in the intestinal tract solution, so that the measured value cannot reflect the real pH value of the intestinal tract. Article published by king pillar et al: "identification of major protease activity in midgut of Heliothis armigera larvae" (journal of insects, Vol.39, No. 1, 2 months 1996), and Zhao Aiping et al: the influence of protease inhibitors on the activity of midgut protease in Grapholitha molesta larvae (proceedings of insects, vol.59, 10, 2016, 1069-1078) was determined by: respectively measuring enzyme activity in a plurality of buffers with different concentration gradients in a pH value range of 5.5-11.0, and determining the pH value with the highest enzyme activity as an optimal pH value; the method has large workload and the measured result can not reflect the enzyme activity under the condition of real intestinal pH value.
Disclosure of Invention
In view of the above technical problems in the background of the present invention, it is an object of the present invention to provide a method for accurately measuring the true pH of the intestinal environment of an insect.
In order to realize the task, the invention adopts the following technical solution:
a method for accurately measuring the real pH value of the intestinal environment of insects is characterized by comprising the following steps:
firstly, taking 30 quick-frozen insect larvae (the number of the quick-frozen insect larvae can be properly adjusted according to the sizes of different types of insect larvae), putting the insect bodies on ice for fixation, quickly cutting off the body walls of the insect larvae along the ventral midline of the insect larvae by using scissors to expose the tissues of the whole digestive tract, cutting off the digestive tract from the front ends of the insect larvae esophagus by using an operation blade, then cutting off the digestive tract at the rear ends of the rear intestines, and then separating out the whole intestinal tracts and the contents of the insect larvae by using tweezers;
step two, placing the dissected intestinal tract and the tissue of the content thereof into a 1.5mL EP tube until the volume of the intestinal tract and the tissue of the content thereof in the EP tube reaches 0.2mL, and then grinding the intestinal tract and the content thereof on ice by using a biological tissue grinder for the EP tube filled with the intestinal tract and the content thereof;
step three, putting the EP tube containing the intestinal tract and the content thereof into a refrigerated centrifuge, and centrifuging for 20min at 4 ℃ and 12000 rpm;
step four, dipping the supernatant in the centrifuged EP tube by using a glass rod, and then, spotting the supernatant on a wide pH test paper, comparing the wide pH test paper with a standard color plate after half a second, reading the pH value, and determining the range of the pH; then, reading out the accurate pH value on a precise pH test paper by the same method; the pH value read out from the precision pH test paper is the real pH value of the intestinal environment of the insect larva.
By adopting the method for rapidly measuring the real pH value of the insect intestinal environment, the real pH value of the insect larva intestinal environment can be accurately obtained in a short time, convenience is provided for the follow-up research of insect intestinal microorganisms and intestinal enzyme systems, and the method has the characteristics of simple operation and real and reliable measured values. Has important guiding value for the food digestion and nutrition acquisition of insects and the development of biological pesticides related to insect midgut.
Detailed Description
In order that the invention may be clearly understood, further details of the invention are set forth in the following description and in the examples.
In this embodiment, the measurement of the real pH value of the intestinal tract of the apple leafroll moth whose host is peach leaf is taken as an example, and it should be noted that the present invention is not limited to this example.
In the following examples, in order to measure the true pH value of the intestinal environment of insects, the inventors collected larvae of healthy 4-year-old apple leaffolders which host peach leaves on the same peach tree, quickly frozen according to a conventional method after collection, then taken out the intestinal tracts of insects, collected a certain amount of intestinal tracts and their contents, ground the intestinal tracts and their contents with a biological tissue grinder, then obtain the supernatant of the intestinal tracts and their contents by high-speed centrifugation, and then measure the true pH value of the intestinal tracts.
The specific operation steps are as follows:
1. taking 30 quick-frozen larvae of apple leaf roller, and cooling and fixing the larvae on an ice bath.
2. Cutting off the body wall of the larvae of the apple leaf roller along the ventral midline of the larvae of the apple leaf roller by using scissors on ice to expose the tissues of the whole digestive tract, cutting off the digestive tract from the front end of the esophagus of the larvae of the apple leaf roller by using an operation blade, cutting off the digestive tract at the rear end of the rear intestine, and separating out the whole intestinal tract and the contents of the larvae of the apple leaf roller by using tweezers.
3. The dissected intestinal tract and its content tissues were immediately put into a 1.5mL EP tube until the volume of the intestinal tract of the larvae and its content tissues in the EP tube reached 0.2mL, and then the intestinal tract and its content tissues were ground on ice using a biological tissue grinder.
4. The EP tube containing the intestinal tract and its contents is placed in a refrigerated centrifuge and centrifuged at 12000rpm for 20min at 4 ℃.
5. The centrifuged clear solution from the EP tube was dipped with a glass rod, spotted on a wide pH paper, compared to a standard color plate after half a second, and read to pH 7.0, determined to be between pH 6.0 and 8.0, and then read to exact pH 6.5 on a precision pH paper using the same method. The pH value read out from the precision pH test paper is the real pH value of the intestinal environment of the larvae of the apple leafroller with the hosts being peach leaves.
Of course, the number and size of the larvae can be adjusted as appropriate for different types of insects.
Claims (1)
1. A method for accurately measuring the real pH value of the intestinal environment of insects is characterized by comprising the following steps:
taking 30 quick-frozen apple leaf roller moth larvae, fixing the larvae on ice, quickly cutting off body walls along the ventral midline of the insect larvae by using scissors to expose the tissues of the whole digestive tract, cutting off the digestive tract from the front end of the esophagus of the apple leaf roller moth larvae by using an operation blade, cutting off the digestive tract at the rear end of the rear intestine, and separating out the whole intestinal tract and the contents of the insect larvae by using tweezers;
step two, placing the dissected intestinal tract and the tissue of the content thereof into a 1.5mL EP tube until the volume of the intestinal tract and the tissue of the content thereof in the EP tube reaches 0.2mL, and then grinding the intestinal tract and the content thereof on ice by using a biological tissue grinder for the EP tube filled with the intestinal tract and the content thereof;
step three, putting the EP tube containing the intestinal tract and the content thereof into a refrigerated centrifuge, and centrifuging for 20min at 4 ℃ and 12000 rpm;
step four, dipping the supernatant in the centrifuged EP tube by using a glass rod, and then, spotting the supernatant on a wide pH test paper, comparing the wide pH test paper with a standard color plate after half a second, reading the pH value, and determining the range of the pH; then, reading out the accurate pH value on a precise pH test paper by the same method; the pH value read out from the precision pH test paper is the real pH value of the intestinal environment of the apple leaf roller moth larva.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103060261B (en) * | 2012-12-29 | 2013-12-04 | 中国农业科学院植物保护研究所 | Method for large-scale quick separation of insect peritrophic membranes |
| CN107063799A (en) * | 2017-04-24 | 2017-08-18 | 中国农业科学院棉花研究所 | The fast acquiring method of intestines in one Species of Lepidopterous Larvae |
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