Method for deducing death time based on corpse fly pupa shell weathering rule and application

Abstract

The invention relates to a method for deducing death time based on a corpse fly pupa shell weathering rule and application, and belongs to the technical field of biology. According to the method, by measuring the contents of C, O and N elements in corpse fly pupa shells at different weathering times, a fitting equation of different functional group contents of the three elements and the weathering time of the pupa shells is constructed, and the functional group contents and the weathering time of the pupa shells are coupled; the weathering time of the corpse fly pupa shell can be deduced only by measuring the contents of different functional groups in the to-be-detected pupa shell, and then the death time is deduced according to the weathering time of the corpse fly pupa shell. The three measured elements C, O and N are regularly changed along with the increase of the weathering time, so that the weathering time can be better represented. The deduction method is simple and efficient, the death time after adult eclosion can be quickly and accurately deduced, the death time deduction time window range of forensic entomology at the present stage is further expanded, and a technical guarantee is provided for policemen to detect a killer's proposal.

Description

Technical Field

[0001] The present invention relates to the technical field of forensic identification, and particularly to a method and application for inferring the time of death based on the weathering law of pupal cases of necrophagous flies. Background Art

[0002] In the detection of criminal homicide cases, the time of death is one of the main bases for delimiting the scope of the case, determining the investigation direction, formulating the investigation plan, locking in the suspect, and excluding the possibility of committing the crime, and it is the starting point for solving the case. Accurately inferring the time of death also helps to save a large amount of police force, material resources, financial resources and time. For hundreds of years since the birth of forensic medicine, the inference of the time of death has always been a research difficulty and hotspot in forensic medicine. Related technologies in forensic entomology have been proven by cases to be practical methods for inferring the time of death of decomposed corpses.

[0003] One of the theoretical bases for using forensic entomology to infer the time of death is to infer the time of death through the growth and development law of necrophagous flies. Most types of necrophagous insects use the corpse as a place for offspring reproduction. From the moment they lay eggs, the "biological clock" for recording the time of death starts. Necrophagous flies can reach the corpse quickly in most cases. Therefore, the age of the offspring of necrophagous insects is infinitely close to the time of death of the corpse. Forensic entomology researchers usually use the growth and development law of the immature stage of necrophagous insects (i.e., the immature stage of insects, including eggs, larvae and pupae), the change in the expression level of specific genes, and the change in the composition of epidermal hydrocarbons to infer the age, so as to provide scientific indicators for inferring the time of death. However, when necrophagous insects complete one generation of development and emerge as adults from the pupal cases, it becomes very difficult to infer the time of death, because currently there is no effective method in forensic entomology to infer the time after emergence.

[0004] Currently, the following are the main methods for inferring the age of Diptera insects after emergence:

[0005] (1) Using adult germ cells to infer the age, but both male sperm and female oocytes will show fluctuations with the hormone levels of adults and are not suitable for use in age inference.

[0006] (2) Using the adult cuticular growth layer for inference. The adult cuticular growth layer is located between the exoskeleton and the cuticle of the insect. It is very difficult for staff without professional systematic learning to observe this part, and currently the research on the adult cuticular growth layer after emergence is not deep enough. Many variables in the existing research have not been fully considered. Most importantly, adults need to continue to develop for a period of time after emergence before their cuticular growth layer can be observed, which makes it impossible to accurately infer the age of adults in the early stage of emergence using this method.

[0007] (3) Using adult wing defects. Adult wing defects have been widely studied, and some scholars have even classified the number, length, and percentage of wing defects. However, environmental factors have a greater impact on wing defects. Therefore, some researchers believe that wing defects can only be used as a tool to confirm the source of flies, because flies from the wild have larger wing defect areas.

[0008] In addition, some biochemical methods are also used to infer the age of Diptera adults after emergence, such as using the changes in adult cuticular hydrocarbons, pteridine levels, and gene and protein expression levels to infer the age of adults. However, the above methods must be used indoors or under closed conditions when the oldest fly adults are captured, and the adults in indoor or closed conditions will die quickly due to lack of food and water, so the relevant methods have great limitations. When the corpse appears in an open environment, the emerged adults will quickly leave the corpse, making the relevant methods inapplicable. In summary, forensic entomology urgently needs to develop a new technology to solve the problems of existing technologies. Summary of the invention

[0009] Therefore, the technical problem to be solved by the present invention is to overcome the problem in the prior art that there is a lack of an effective method for inferring the time of death based on sarcophagous flies.

[0010] To solve the above technical problems, the present invention provides a method and application for inferring the time of death based on the weathering law of sarcophagous fly pupae. The present invention measures the contents of three elements, C, O and N, in sarcophagous fly pupae with different weathering times, constructs a fitting equation of different functional group contents of the three elements and the weathering time of the pupae, and couples the functional group content with the weathering time of the pupae. Therefore, it is only necessary to measure the functional group content in the pupae to be tested to infer the weathering time of the sarcophagous fly pupae, and then infer the time of death according to the weathering time of the sarcophagous fly pupae. The present invention selects pupae as the sample to be tested, and the efficiency of collecting these pupae is higher than that of capturing active adults. And the captured adults need to identify their source (developed from larvae on the corpse or flying in the wild), and collecting pupae greatly saves this part of time and energy consumption. The three elements of C, O and N measured by the present invention show regular changes with the increase of weathering time, so they can better characterize the weathering time. The inference method of the present invention is simple and efficient, and can realize the rapid and accurate inference of the time of death after the adult insects emerge, further expanding the time window range of the time of death inference in the current forensic entomology, and providing technical support for the police to solve homicide cases.

[0011] The first object of the present invention is to provide a method for inferring the weathering time based on the pupa shells of sarcophagous flies, comprising the following steps:

[0012] S1. Cultivate carrion flies until they pupate. Take out the pupae and cultivate them until they emerge as adults. Take out the pupal cases left after the pupae emerge as adults and store them at a constant temperature. Take out the pupal cases at preset intervals and freeze-dry them to obtain freeze-dried pupal cases with different weathering times.

[0013] S2. Measure the contents of C1s functional groups, O1s functional groups, and nitrogen element in the freeze-dried pupal cases respectively, and establish fitting curves of the contents of C1s functional groups, O1s functional groups, and nitrogen element at different weathering times of the carrion fly pupal cases.

[0014] S3. Measure the contents of the C1s functional groups, O1s functional groups, and nitrogen element in the sample to be tested, and substitute them into the fitting curves in S2 to infer the weathering time of the sample to be tested.

[0015] Among them, the C1s functional groups are selected from one or more of C-N-C=O, C-NH 2 , O=C-NH 2 , C-C=O, and the O1s functional group is C=O.

[0016] Further, the carrion flies include Lucilia sericata. The present invention selects Lucilia sericata as the sample. Lucilia sericata is widely distributed and has strong survival ability. Its pupal case is the residue left after Lucilia sericata emerges as an adult and can exist at the corpse scene for a long time. Measuring the composition in the pupal case of Lucilia sericata and observing the change law of the composition at different time points under constant temperature conditions is of great significance for broadening the application scope of forensic entomology.

[0017] Further, the temperature for weathering preservation in step S1 is 20 - 30 °C.

[0018] Further, the process of cultivating the carrion flies to pupae in step S1 is: raise and collect the eggs of the carrion flies, and raise the eggs at a constant temperature until they pupate.

[0019] Further, the temperature for constant-temperature raising is 20 - 30 °C.

[0020] Further, the preset time is 1 - 10 days.

[0021] Furthermore, the contents of C1s functional groups, O1s functional groups, and nitrogen elements in freeze-dried pupa cases were measured using X-ray photoelectron spectroscopy (XPS). XPS was selected for the measurement in this invention. XPS is a technique that analyzes samples by using the photoelectric effect to influence the vibration of the extranuclear electrons of the samples to be measured. It is currently widely used in chemistry and related fields and is an ideal method for measuring surface chemical composition and chemical state. It can also provide information such as composition content and chemical bond formation. Therefore, in this invention, XPS was used to measure the functional group composition in the pupa cases of Lucilia sericata, to find components with potential age inference effects, and to provide new ideas for solving cases where necrophagous flies with eclosion appear.

[0022] The second object of the present invention is to provide an application of the above method in inferring the time of death.

[0023] Furthermore, the application is as follows: Collect the pupa cases of necrophagous flies on the corpse and detect the contents of C1s functional groups, O1s functional groups, and nitrogen elements in the pupa cases, and substitute them into the fitting equations of the contents of C1s functional groups, O1s functional groups, and nitrogen elements at different weathering times of the pupa cases of necrophagous flies to infer the weathering time of the necrophagous flies. Substitute the obtained weathering time into the equation of the time of death, so as to infer the time of death of the corpse; where the equation of the time of death is: Time of death = Time from the arrival of necrophagous flies at the corpse to oviposition and eclosion + Weathering time. When the environmental temperature is about 25 °C, taking Lucilia sericata as an example, the time from the arrival of this species at the corpse to oviposition and eclosion is 335.63 ± 19.33 hours.

[0024] The third object of the present invention is to provide a kit for identifying the eclosion time of pupa cases of necrophagous flies. The kit contains reagents for detecting the contents of C1s functional groups, O1s functional groups, and nitrogen elements in the pupa cases of necrophagous flies. Among them, the C1s functional groups are selected from one or more of C-N-C=O, C-NH 2 , O=C-NH 2 , C-C=O, and the O1s functional group is C=O.

[0025] The fourth object of the present invention is to provide an application of the above kit in inferring the time of death.

[0026] The beneficial effects of the present invention:

[0027] The present invention provides a method and application for inferring the time of death based on the weathering law of the pupal cases of sarcophagous flies. By measuring the contents of three elements, namely C, O, and N, in the pupal cases of sarcophagous flies at different weathering times, the present invention constructs fitting equations for the contents of different functional groups of the three elements and the weathering time of the pupal cases, coupling the content of the functional groups with the weathering time of the pupal cases. Therefore, it is only necessary to measure the content of the functional groups in the to-be-detected pupal cases to infer the weathering time of the pupal cases of sarcophagous flies, and further infer the time of death based on the weathering time of the pupal cases of sarcophagous flies. The present invention selects pupal cases as the to-be-detected samples. Compared with capturing active adults, the efficiency of collecting pupal cases is higher. And the captured adults need to identify their sources (developed from larvae on the corpse or flown from the wild), and collecting pupal cases greatly saves the time and energy consumption in this part. The three types of functional groups selected by the present invention show regular changes with the increase of the weathering time, so they can better characterize the weathering time. The inference method of the present invention is simple and efficient, and can quickly and accurately infer the time of death after adult emergence, further expanding the time window range of the time-of-death inference in the current forensic entomology, and providing technical support for the police to solve homicide cases. Description of the Drawings

[0028] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to the specific embodiments of the present invention and in combination with the drawings, wherein

[0029] Figure 1 is the XPS full spectrum of the empty pupal case of Lucilia sericata, where the X-axis is the binding energy and the Y-axis is the absorption peak of the atom at this binding energy. The XPS full spectrum of the empty pupal case of Lucilia sericata consists of three main peaks;

[0030] Figure 2 is the C1s fine spectrum of the empty pupal case of Lucilia sericata from 0d to 15d;

[0031] Figure 3 is the C1s fine spectrum of the empty pupal case of Lucilia sericata from 20d to 30d;

[0032] Figure 4 is the O1s fine spectrum of the empty pupal case of Lucilia sericata from 0d to 20d;

[0033] Figure 5 is the O1s fine spectrum of the empty pupal case of Lucilia sericata from 25d to 30d;

[0034] Figure 6 is the graph of the change law of different components of the empty pupal case of Lucilia sericata with time;

[0035] Figure 7 is the content change trend of TypeⅡ C1s, TypeⅢ C1s, N1s, and TypeⅠ O1s. Detailed Embodiments

[0036] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the embodiments cited do not limit the present invention.

[0037] Example 1

[0038] Put 20 g of fresh lean pork into the breeding cage, collect the eggs within one hour (N > 500), put them into the breeding box, and breed them at a constant temperature with fresh lean pork. After pupation, pick out the pupae and put them into separate petri dishes, then put them back into the incubator and continue to breed until eclosion.

[0039] Pick out the pupal cases that have eclosed within 12 h and put them into a constant temperature breeding box at 25 °C for preservation. Take 3 pupal cases each at 0, 5, 10, 15, 20, 25, and 30 days, wash them clean with an ultrasonic cleaner, drain the water, and store them at -80 °C.

[0040] Freeze-dry the samples in a freeze-dryer for 48 h, then put them into EP tubes, seal them with sealing film, store them at -80 °C, and measure the samples within one week. The steps are as follows:

[0041] 1. Cut off the upper 1 / 3 of the pupal case with sterile scissors and unfold it.

[0042] 2. Stick the unfolded pupal case on the conductive adhesive.

[0043] 3. Put the sample on the sample stage of the instrument (America Thermo ESCALAB 250XL) to test the outside of the pupal case. Monochromatic Al Ka (hv = 1486.6 eV), power 150 W, 400 μm beam spot.

[0044] 4. Use Avantage to correct the data with C1s = 284.8 eV, perform peak fitting and elemental semi-quantitative processing. The data after peak fitting is imported into Origin 2021 for plotting, and the elemental change trend graph is drawn with Origin2021.

[0045] 5. Data analysis and inference method

[0046] According to the XPS full spectrum ( Figure 1 ), the analysis shows that the outer layer of the pupal case of Lucilia sericata is composed of three elements: C (284.8 eV), N (401.32 eV), and O (531.84 eV). The C1s peak fitting results show that the C1s in the empty pupal case of Lucilia sericata after eclosion is mainly composed of three states: 1) Type Ⅰ C1s: a carbon atom is connected to a hydrogen atom or another carbon atom (C-C or C-H); 2) Type Ⅱ C1s: the carbon atom in the amide bond is connected to a nitrogen atom and then in series with a carbonyl group or a carbon atom is connected to an amino group (C-N-C=O or C-NH 2)); 3) Type Ⅲ C1s: The carbon atom is connected to an amide bond or a carbon-oxygen double bond (O=C-NH 2 or C-C=O) in three conformational states. O1s is composed of C=O (Type Ⅰ O1s) and C-O (Type Ⅱ O1s), and their binding energy absorption peaks are 532.1 eV and 534.4 eV respectively, and some of their contents show regular changes over time.

[0047] From the analysis results of this data ( Figure 2 and Figure 3 ), it is known that the content of Type Ⅰ C1s shows irregular fluctuations at a low level; the content of Type Ⅱ C1s first decreases, then increases and then decreases, reaching a trough at about 15 days and a peak at 20 days, and then continues to decline; the content of Type Ⅲ C1s shows a trend of first increasing and then decreasing, reaching a peak at about the 15th day.

[0048] From the analysis results of this data ( Figure 4 and Figure 5 ), it is obtained that Type Ⅰ O1s first increases and then decreases at a relatively low content, reaching a peak at 15 days; the content of Type Ⅱ O1s is relatively stable.

[0049] The XPS elemental semi-quantitative analysis results show that ( Figure 6 ), the content of Type Ⅰ C1s shows irregular fluctuations at a low level; the content of Type Ⅱ C1s first decreases, then increases and then decreases; the content of Type Ⅲ C1s shows a trend of first increasing and then decreasing; the content of N1s first increases and then decreases. Type Ⅰ O1s first increases and then decreases at a relatively low content; the content of Type Ⅱ O1s is relatively stable.

[0050] Therefore, we can infer the post-emergence time of Lucilia sericata according to the characteristics that the contents of Type Ⅱ C1s, Type Ⅲ C1s, N1s, and Type Ⅰ O1s change regularly over time.

[0051] Table 1 Simulation equations for the change of relative element content (%) with the number of days after emergence (d)

[0052] Element Equation <![CDATA[R 2 > N1s <![CDATA[y = 0.0031x 3 - 0.1817x 2 + 2.8055x + 13.243]]> 0.963 TypeIIC1s <![CDATA[y = -0.0057x 3 + 0.2917x 2 - 4.0408x + 24.696]]> 0.771 TypeIIIC1s <![CDATA[y = 0.0028x 3 - 0.1127x 2 + 1.2895x + 3.7444]]> 0.855 TypeIO1s <![CDATA[y = 0.0014x 3 - 0.0957x 2 + 1.5781x + 3.0673]]> 0.798

[0053] From the analysis results of this ( Figure 7)It can be seen that it is divided into four time stages: The first stage is approximately from 0 to 10 days. At this time, the trend of N1s is increasing, the trend of TypeⅡC1s is decreasing, and the trends of TypeⅢC1s and TypeⅠO1s are increasing; The second stage is approximately from 10 to 20 days. At this time, the trends of N1s, TypeⅢC1s and TypeⅠO1s are decreasing, and the trend of TypeⅡC1s is increasing; The third stage is approximately from 20 to 25 days. The trends of N1s and TypeⅠO1s are decreasing, and the trends of TypeⅡC1s and TypeⅢC1s are increasing; The fourth stage is approximately from 25 to 35 days. At this time, the trends of N1s, TypeⅡC1s and TypeⅠO1s are decreasing, while the trend of TypeⅢC1s is increasing.

[0054] The inference method is as follows: Take the data of 3 groups of N1s, TypeⅡC1s, TypeⅢC1s, and TypeⅠO1s at consecutive and close times. If it is judged that the trend of N1s is increasing, the trend of TypeⅡC1s is decreasing, and the trends of TypeⅢC1s and TypeⅠO1s are increasing, then it can be inferred that the time after emergence of Lucilia sericata angustifrons is from 0 to 10 days.

[0055] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of this invention.

Claims

1. A method for estimating weathering time based on pupa shells of sarcophagous flies, characterized in that: The following steps are involved: S1, culturing sarcophagous flies to pupae, taking out the pupae and culturing them until they emerge, taking out the pupal shells left after the pupae emerge and storing them at a constant temperature, taking out the pupal shells at preset time intervals, and obtaining pupal shells with different weathering times; S2. Determine the contents of C1s functional group, O1s functional group and nitrogen in the pupal shell respectively, and establish fitting curves between the contents of C1s functional group, O1s functional group and nitrogen and the weathering time of the pupal shell of sarcophagous flies; S3, determining the contents of the C1s functional group, the O1s functional group and the nitrogen element in the sample to be tested, and substituting them into the fitting curve of S2 to infer the weathering time of the sample to be tested; Wherein, the C1s functional group is selected from one or more of CNC=O, C-NH2, O=C-NH2, CC=O, and the O1s functional group is C=O.

2. The method according to claim 1, characterized in that The sarcophagous flies include Lucilia tenuifolia.

3. The method according to claim 1, characterized in that: The constant temperature storage in step S1 is 20-30°C.

4. The method according to claim 1, characterized in that: The process of culturing the sarcophagous flies to pupae in step S1 is: raising the sarcophagous flies and collecting their eggs, and raising the eggs at a constant temperature until they pupate.

5. The method according to claim 4, characterized in that The temperature of the constant temperature breeding is 20-30°C.

6. The method according to claim 1, characterized in that The preset time is 1-10 days.

7. The method according to claim 1, characterized in that The contents of C1s functional group, O1s functional group and nitrogen element in pupa shell were determined by X-ray photoelectron spectroscopy.

8. Use of the method according to any one of claims 1 to 7 in estimating the time of death.

9. A kit for identifying the emergence time of pupa of sarcophagous flies, characterized in that: The kit contains reagents for detecting C1s functional group, O1s functional group and nitrogen element in sarcophagous fly pupae, wherein the C1s functional group is selected from one or more of CNC=O, C-NH2, O=C-NH2, CC=O, and the O1s functional group is C=O.

10. Use of the kit according to claim 9 in estimating time of death.

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