TWI660078B - Ant species analyzing method by pangolin feces - Google Patents

Abstract

The invention provides a method for sieve-washing and analyzing ant species from a pangolin waste, which comprises the following steps: drying the waste: collecting the waste of the pangolin, and removing the moisture in the waste by a drying method; sieving the insect remains: The dried waste is immersed in water to form an analysis solution, and a bubble generation method is used to spray fine bubbles in the analysis solution, and the insect remains in the analysis solution are adsorbed on the surface of the bubbles, and floated to the water surface for several screenings Filtration; Filtration: After sieving and filtering, the water and impurities in the analysis solution are separated to retain the insect remains in the analysis solution; the insect remains are dried; and macro photography is taken to identify and identify the species and quantity: Sampling and analysis to determine the pangolin find Anteater species and count foraging.

Description

Method for sieve washing and analysis of ant species from pangolin excrement

The invention relates to a method for analyzing the pangolin's feeding habits, and in particular to a method for sieve-washing and analyzing ant species for self-pangolin excretion of pangolin foraging habits.

In order to understand the food requirements of mammals in the field of food ecology of wild animals, non-invasive stool analysis methods are mainly used in food research to avoid disturbing the habits of wild animals.

In the study of anteater mammal ecology, the feeding habits of anteater mammals are usually investigated by the contents and excretion of the stomach. Among them, ant mammals usually eat ants and termites as their main food. The analysis of foraging habits is to identify the species of foraging ants and termites based on the appearance characteristics of ants and termites that have not been completely digested (hereinafter, refer to Non-Patent Document 1, Non-Patent Document 2).

However, the process of fecal analysis is time consuming and the accuracy of identifying ant remains is low. Specifically, the excreta contains more than 50% of impurities (such as soil, sand, and plant matter) (hereinafter, refer to Non-Patent Document 3), and sampling and analysis are not performed through filtration. It is easy to fail because there are too many impurities. Effectively identify and ignore small insect remains, which affects the accuracy and reproducibility of ant-eater mammals in foraging ant species.

In addition, some ecological researchers believe that during the sieve washing process, some insect debris may be filtered out, and termite and some ant debris may be lost, which may affect the accuracy of analyzing ants and termite species. [Prior Art Literature] [Non-Patent Literature 1]: Stewart et al. 1999. Ecological factors affecting the feeding behavior of pangolins ( Manis temminckii ). Journal of Zoology 247: 281-292. [Non-Patent Literature 2]: Taylor et al 2002. The feeding ecology of the aardvark Orycteropus afer. Journal of Arid Environments 50: 135–152. [Non-Patent Document 3]: Mahmood et al. 2013. Plant species association, burrow characteristics and the diet of the Indian pangolin, Manis crassicaudata , in the Potohar Plateau, Pakistan. Pakistan Journal of Zoology 45: 1533-1539.

In order to solve the above-mentioned problem, the present invention provides a method for sieve washing and analyzing ant species from a pangolin excrement, which can determine the species of pangolin foraging ants and calculate the number of foraging.

In order to solve the above object, an embodiment of the present invention provides a method for sieve washing and analysis of ant species from a pangolin waste, which comprises the following steps: collecting the waste of the pangolin, and removing the moisture in the waste by a drying method ; Sift and wash the insect remains: immerse the dried waste in water, and evenly stir and separate the waste to form an analysis solution, and use a bubble generation method to spray fine bubbles in the analysis solution to make the insect remains in the analysis solution Adsorbed on the surface of air bubbles and floated on the water surface for several times to filter. Filtering: After filtering and filtering, filter the water and insect debris in the analysis solution to remove the insect debris in the analysis solution; and macro photography shooting and Identification of species and quantity: The insect remains are dried, and the species of pangolin foraging ants are judged and the foraging quantity is calculated.

In this way, the method of the present invention can effectively identify the vast majority of insect debris in pangolin waste, and for anteater mammals with a large amount of waste, the present invention is very time-saving in the processing process, and in the analysis and It has both an accuracy rate and a recognition rate of up to 90% in terms of calculations, and it has improved the efficiency of research in the field of ecological studies of pangolins and ants.

In order to facilitate the description of the central idea of the present invention in the above-mentioned summary of content, specific embodiments are described below. Various objects in the embodiments are drawn according to the proportions suitable for enumeration, rather than the proportions of the actual components, which will be described beforehand.

Please refer to FIG. 1 to FIG. 4. The present invention provides a method for sieve washing and analysis of ant species from a pangolin waste, which comprises the following steps:

Dry excrement S1: Researchers can first collect the pangolin's excrement in a temperature of minus 20 ° C, and avoid the spoilage and deterioration of the excrement. For research and analysis, defrost the frozen excrement directly, and then thaw it. The waste is placed in a drying method at 60 ° C to remove the moisture in the waste. In the embodiment of the present invention, the drying method is to dry the waste in an oven environment at 60 ° C.

Sifting the insect remains S2: Next, the dried waste is immersed in water, wherein the waste is immersed in 200 ml of hot water and stirred uniformly for 10 minutes to separate and crush the waste to form an analytical solution; subsequently, Fine bubbles are sprayed into the analysis solution by a bubble generation means, so that the insect remains in the analysis solution are adsorbed on the surface of the bubbles.

Specifically, the bubble generation method of the present invention uses a sprayer to spray a water column on the analysis solution. In the process of water column spray, water injection will simultaneously inject the air in the environment into the analysis solution, thereby generating fine bubbles in the analysis solution. At this time, the water column will drive the analysis solution to circulate, and the bubbles will float toward the water surface, which will disturb the insect debris in the analysis solution, which will be adsorbed on the surface of the bubbles and float on the water surface. The water surface was filtered several times.

Filtering S3: After filtering, the analysis solution was filtered through a filter to remove water and remaining large insect debris, and directly extracted and preserved with tweezers.

Macro photography shooting and identification of species and quantity S4: Dry the stored insect remains to remove and dry excess water, and sample the dried insect remains for macro photography shooting and identification of species and quantity to determine pangolin foraging The species of ants, and the number of pangolin foraging ant species is calculated using a hemocytometer. In the embodiment of the present invention, the pangolin foraging ant species is determined using the incompletely digested ant head shell and termite big jaws as indicators.

In this way, the present invention uses a bubble screening method to remove most of the insect debris in the excrement, and then has an accuracy and recognition rate of up to 90% in both analysis and calculation. Moreover, the present invention is very time-saving in processing, In the field of diet research, it has the effect of improving research efficiency.

The following examples and comparative examples will provide a person with ordinary knowledge in the technical field with a complete disclosure and description of articles, devices and / or methods of patent application made and evaluated here, which are merely examples and are not intended to limit the present invention. Category.

The experimental samples of the present invention are from 2010 to 2016. Fresh excrements of 132 pangolins were taken in the field, and each excrement sample was labeled and stored separately and stored in a negative 20 ° C environment. During the experimental analysis, 4 exclusion codes were randomly selected from 132 exclusion samples for experiments. Among them, the exclusion codes extracted by the experiment of the present invention are 20120106, 20150220, 20140407, and 20141015, and then the 4 codes are ranked. The remains were placed in an oven at 60 ° C., dried and thawed for 24 hours, and then the excretion after drying was compared with the comparative example and the experimental comparison of the examples. Comparative example

The 4 encoded dried residues are not filtered (not passed through the processes of 0021, 0022, 0023), and each is directly and uniformly arranged in a rectangular plastic dish, and a small amount of 90% alcohol is added to make the dried Spread the waste as evenly as possible on the plastic disc. Examples

Fill the four encoded dried excrements into beakers, and fill each beaker with 200 ml of hot water, and stir uniformly for 10 minutes to separate and crush the waste to form an analysis solution.

Subsequently, a sprayer is used to spray the water column on the analysis solution. During the water column spray process, water injection will simultaneously inject air from the environment into the analysis solution, causing fine bubbles to form in the analysis solution. At this time, the water column will drive the analysis solution to circulate. In addition, the bubbles will float toward the water surface, which will disturb the insect debris in the analysis solution, which will be adsorbed on the surface of the bubbles and float on the water surface. The researchers used the filter to filter the water on the water surface of the analysis solution 8 to 10 times. This filters most of the impurities in the waste.

Next, the filtered analysis solution is filtered, and the water and large insects are filtered out by a filter and extracted with tweezers. Finally, the stored insect debris is dried to remove and dry excess water, and the dried insects are dried. The wreckage was sampled for analysis and observation.

The analysis of the experimental results and the verification of the characteristics of the examples and comparative examples are discussed below: Analysis of the number of ant species in the examples and comparative examples

The present invention judges the way of pangolin foraging for ant species: in the examples and comparative examples, macroscopic photography is used to identify and identify species and quantity photos to observe digestive debris. Because the ant's head shell is not easily digested, it is incompletely digested. The ant's head and shell are used as indicators to determine the species of ants, while the termites use the big jaws as indicators to identify the species and count the numbers.

The examples and comparative examples are used to calculate the number of ant species, and the calculation method is to use a blood cell counter to calculate, and use the following formula to convert the identifiable number into a percentage:

Recognizable termite jaw percentage (%) = M1 / M2 * 100%

Recognizable ant head shell percentage (%) = A1 / A2 * 100%

Among them, M1 = the number of recognizable termite objects, M2 = the maximum number of recognizable termite species in each comparative example; A1 = the number of recognizable ant head shells, A2 = recognizable ant species in each comparative example The maximum number.

In addition, because the composition of each excrement is different, the number of scattered areas of the digested ant remains is not equal. Therefore, a 1-gram sample was taken in the comparative example and the example to calculate the maximum area required for the dispersion of identifiable ant remains . Table 1, Comparative Examples and Examples, the maximum area required to spread 1 gram of wasteland Exclusion Sample Code 20120106 20150220 20140407 20141015 Comparative example (disc / area) 16 (720cm ² ) 16 (720cm ² ) 32 (1,420cm ² ) 32 (1,420cm ² ) Example (disc / area) 2 (90cm ² ) 2 (90cm ² ) 9 (405cm ² ) 8 (360cm ² )

As shown in Table 1, the area required to spread the recognizable ant debris in the examples is significantly smaller than that in the comparative example, and with reference to FIG. 2, each copy is left in a different spread area (1-32 plates ) Respectively, take macro photography photos (in units of 1.5 cm ² ), and compare the density differences of ant debris under different flat areas. Among them, the images indicate A, B, C, D, E, and F respectively. Screening washed excrement samples, changes in insect debris density in different flat areas (1,2,4,8,16,32 plastic trays). The image label G indicates a photograph of the wreckage taken after sieving and filtering.

Next, the numbers of termite species and ant species in the examples and comparative examples are compared. Table 2. Each gram of dried pangolin was left under the methods of the comparative examples and the examples. The number of termites and ant species identified was compared with the table, showing that there was no significant difference in the types and numbers identified by the two methods. Ant taxa Mean ± SD (comparative example) Mean ± SD (Example) t p value Taxon ISOPTERA Termitidae Odontotermes formosanus LM (large worker) 52.75 ± 44.81 54.25 ± 50.51 -0.37 0.73 RM (large worker) 59.00 ± 51.95 60.75 ± 53.64 -0.51 0.65 LM (minor worker) 54.25 ± 62.62 55.00 ± 67.88 -0.18 0.87 RM (minor worker) 58.00 ± 65.49 62.50 ± 78.73 -0.61 0.58 LM (soldier) 14.75 ± 17.15 14.50 ± 14.66 0.17 0.88 RM (soldier) 20.75 ± 19.57 20.00 ± 24.81 0.23 0.84 LM (alate) 0.25 ± 0.50 0.50 ± 1.00 -1.00 0.39 Pericapritermes nitobei LM (worker) 2.25 ± 4.50 2.00 ± 4.00 1.00 0.39 RM (worker) 2.00 ± 3.37 0.75 ± 1.50 1.32 0.28 Nasutitermes parvonasutus LM (worker) 18.75 ± 31.19 6.50 ± 10.50 1.18 0.32 RM (worker) 20.25 ± 31.31 10.50 ± 17.82 1.43 0.25 Rostrum (Soldier) 3.25 ± 3.95 1.25 ± 1.50 1.41 0.25 HYMENOPTERA Myrmicinae Pheidologeton yanoi Minor worker 24.25 ± 47.17 21.75 ± 42.18 0.99 0.40 Minor soldier 175.50 ± 351.00 172.25 ± 344.50 1.00 0.39 Pheidole nodus Worker 55.50 ± 86.97 52.00 ± 81.78 1.35 0.27 Soldier 9.00 ± 10.52 9.25 ± 10.75 -1.00 0.39 Pheidole fervens Worker 77.75 ± 143.68 76.50 ± 142.47 1.67 0.19 Soldier 7.00 ± 12.08 6.50 ± 11.09 1.00 0.39 Pheidole parva Worker 4.50 ± 8.35 4.00 ± 6.73 0.58 0.60 Crematogaster schimmeri Worker 32.75 ± 57.78 25.50 ± 44.58 1.10 0.35 Crematogaster nawai Worker 9.00 ± 18.00 6.50 ± 13.00 1.00 0.39 Crematogaster sp. Worker 31.00 ± 62.00 31.25 ± 62.50 -1.00 0.39 Rhoptromyrmex wroughtonii Worker 0.50 ± 1.00 0.75 ± 1.50 1.63 0.20 Formicinae Camponotus monju Minor worker 89.25 ± 162.89 87.25 ± 160.81 1.63 0.20 Pseudolasius binghami taivanae Worker 7.25 ± 13.84 7.75 ± 15.50 -0.58 0.60 Anoplolepis gracilipes Worker 11.00 ± 16.55 7.25 ± 13.20 1.72 0.18 Lepisiota rothneyi wroughtonii Worker 7.25 ± 14.50 6.50 ± 13.00 1.00 0.39 Ponereinae Leptogenys kitteli Worker 1.25 ± 1.50 0.75 ± 0.96 1.73 0.18 Aenictinae Aenictus ceylonicus Worker 62.75 ± 95.33 56.5 ± 84.79 1.18 0.32 Pupae 1.75 ± 3.50 1.50 ± 3.00 1.00 0.39 No. of termite species identified 2.00 ± 1.15 1.75 ± 0.96 1.00 0.39 No. of ant species identified 8.00 ± 5.35 7.75 ± 5.62 1.00 0.39

As shown in Table 2, the number of ant species identified in the examples and comparative examples was the same, and there were 3 types of termites and 17 types of ants, respectively. Examples and Comparative Examples

Record the time spent on various processes (such as sample flattening, macro photography, filtering, and species identification) in different processes. In the experiment of the present invention, the time consumption of each fecal sample is calculated by the following formula: Comparative example: each scattered consumption time = ST + PT + IT Example: consumption time = FT + ST + PT + IT where ST = Flattening time / Flattening time per plastic disc * Number of plastic discs PT = Photography time / Plastic disc shooting time * Number of plastic discs IT = Identification time / Identification time of each plastic disc * Number of plastic discs FT = Filtering Elapsed time of treatment / 1g Dry weight excretion identifies percentage of time spent in ant species

The elapsed time of the embodiment and the comparative example are converted into percentages respectively, and the conversion formula of the present invention is: the percentage of the elapsed time = T1 / T2 * 100%. Among them, T1 = each dispersed elapsed time T2 = each comparative example Time-consuming

The statistical results are shown in Figures 3 and 4. Even though the comparative example has a better species identification result after flattening to 32 trays, it is time consuming. In contrast, the processing time of the embodiment is half as long as that of the comparative example. In addition, the samples of the embodiment can effectively identify and calculate the number and species of termites and ants, and the identification number can reach 90 in the comparative example. %.

In summary, the present invention uses bubble screening to extract insect remains in the exhumation, and then has an accuracy and identification rate of up to 90% in both analysis and calculation; for the application of different anteater mammals, For ant-eater species with a large amount of waste, the present invention will be very time-saving in the treatment process, and it has the effect of improving research efficiency in the field of pangolin and ant-feeding.

Many modifications and changes for those skilled in the art are obvious without departing from the scope and spirit of the present invention. Other embodiments of the present invention are obvious to those skilled in the art from the description of the description and the method of the present invention disclosed herein. The description and examples are merely illustrative, and the true scope and spirit of the present invention will be pointed out in the following patent application scope.

S1‧‧‧Dry excretion

S2‧‧‧ Screening and washing of insect remains

S3‧‧‧Filter

S4‧‧‧ Macro photography and identification of species and quantity

Figure 1 is a flowchart of the method of the present invention. FIG. 2 is an observation analysis diagram of a comparative example of the present invention under a microscope at different magnifications. FIG. 3 is a statistical chart of the time consumption percentage of the examples and comparative examples of the present invention. FIG. 4 is a statistical chart of time consumption percentage according to an embodiment of the present invention.

Claims (6)

A method for sieve-washing and analyzing ant species from a pangolin excrement, which includes the following steps: drying excretion: collecting the excretion of pangolins and removing the moisture in the excretion by a drying method; sieving the insect remains: drying the The excretion is immersed in water, and the excretion is uniformly stirred to separate the excretion to form an analysis solution, and a bubble generation method is used to spray fine bubbles in the analysis solution, so that the insect debris in the analysis solution is adsorbed on the surface of the bubble, and Floating on the water surface for several times of sieve washing and filtering; Filtration: After sieve washing and filtering, filter the water and impurities of the analysis solution through a filter to collect macro insects in the analysis solution that are not brought up by air bubbles. Macro photography And identification of species and quantity: the insect remains are dried, and the dried insect remains are sampled for macro photography to identify ant species that the pangolin seeks for food and calculate the amount of foraging. The method for screening and analyzing ant species from the pangolin wastes as described in claim 1, wherein in the macro photography shooting and identifying the species and quantity, the pangolin finding is determined using the incompletely digested ant head shell as an indicator. Ant-eater species. The method for screening and analyzing ant species from the pangolin wastes as described in claim 1 or 2, wherein in the macro photography shooting and identifying species and quantity steps, the incompletely digested ant's jaw is used as an indicator Determine the species of pangolin foraging termites. The method for screening and analyzing ant species from the pangolin wastes as described in claim 1, wherein, in the macro photography shooting and identifying species and quantity steps, the number of pangolin foraging ant species is calculated using a blood cell counter . The method for sieve-washing and analyzing ant species from the pangolin waste as described in claim 1, wherein, in the step of sieve-washing the insect remains, the bubble generating means sprays a water column on the analysis solution with a sprayer, and the water column It has the fine bubbles mentioned therein. The method for sieve-washing and analyzing ant species from the pangolin excrement described in claim 1, wherein in the drying excretion step, the drying method is to dry the excretion in an oven environment at 60 ° C.