Isolation and Identification of Volatile Substances with Attractive Effects on Wohlfahrtia magnifica from Vagina of Bactrian Camel

Simple Summary Vaginal myiasis is one of the most serious parasitic diseases in Bactrian camels, and the disease has brought serious economic impacts to local herdsmen. Wohlfahrtia magnifica is the main pathogen causing hemorrhagic trauma and myiasis in the natural openings of humans and animals. The study showed that methylheptenone, 1-octen-3-ol, and propyl butyrate can all cause an antennae reaction of Wohlfahrtia magnifica. Moreover, except for propyl butyrate, the higher the concentrations of the other two compounds, the stronger the attractivity to the Wohlfahrtia magnifica, and the mixture of them at the ratio of 1:1 can enhance the attractivity. This study laid a foundation for biological control of vaginal myiasis in Bactrian camels. Abstract Vaginal myiasis is one of the most serious parasitic diseases in Bactrian camels. At present, there are no reports on biological control measures of the disease. In this paper, the metabolomic analysis of vaginal secretions from susceptible and non-susceptible camels was performed by ACQUITY UPLC H-Class Ultra Performance Liquid Chromatograph. The results matched in 140 vaginal compounds. Methylheptenone, 1-octen-3-ol, and propyl butyrate and their mixtures were selected for gas chromatography-electroantennography (GC-EAD), electroantennography (EAG), behavioral experiments and trapping experiments of Wohlfahrtia magnifica (W. magnifica). Results showed that the W. magnifica had EAG responses to the three compounds, respectively. The EAG responses of female flies to different concentrations of methylheptenone were significantly different, but to the others had no significant difference, and there was no significant difference in the same compounds between the different sexes. Behavioral and trapping experiments showed that methylheptenone and 1-octen-3-ol have significant attraction to W. magnifica, but there was no significant difference to propyl butyrate. When methylheptenone and 1-octen-3-ol were mixed in different proportions, it was found that a mixture at the ratio of 1:1 and 0.5:1 had extremely significant and significant attraction, respectively, to both male and female W. magnifica. The study showed that, except for propyl butyrate, the higher the concentrations of the other two compounds, the stronger the attractivity to the W. magnifica, and a mixture at the ratio of 1:1 could enhance the attractivity to the W. magnifica.


Introduction
Bactrian camels are mainly distributed in Asia and surrounding cold desert areas, such as Mongolia, China, Kazakhstan, Russia and so on, and are an important material resource

Liquid Mass Spectrometry Analysis of Vaginal Secretions from Bactrian Camels
The vaginal secretions of Bactrian camels were collected with sterile cotton swabs, where 10 samples, 10 samples, and 6 samples were collected from female camels that recovered after the disease, that were in a period of disease, and that did not suffer from the disease, and were recorded as group1, group2 and group3, respectively. The heads of cotton swabs with samples from each group were placed in a 5 mL centrifuge tube and marked, 1 mL ethyl acetate (Shanghai Maclin Biotechnology Co., LTD, Shanghai, China) was added, and the samples were fully dissolved by continuous oscillations of constant temperature ultrasonator in a water bath for 1 h. The solution was filtered into a sample bottle by inorganic filter and detected by Ultra Performance Liquid Chromatography.
Setting of test parameters of AQUITY UPLC H-Class Ultra Performance Liquid Chromatograph (Waters): ACQUITY UPLC C18 BEH (2.1 mm × 100 mm,1.7 µm, Waters) was selected as the chromatographic column; the column temperature and sample chamber temperature were constant at 40 • C and 10 • C, respectively; mobile phase A was water (0.1% methanoic acid); and mobile B was acetonitrile (0.1% methanoic acid). The test gradient and parameters are shown in the following table (Tables 1 and 2).

GC-EAD of Methylheptenone, 1-octen-3-ol, and Propyl Butyrate
Methylheptenone (Shanghai Yuanye Biotechnology Co., LTD, Shanghai, China), 1octen-3-ol (Shanghai Yuanye Biotechnology Co., LTD, Shanghai, China) and propyl butyrate (Shanghai Maclin Biotechnology Co., LTD, Shanghai, China) were prepared into 10 −2 µg/µL samples with n-hexane (Shanghai Maclin Biotechnology Co., LTD, Shanghai, China), respectively. The antennae of the W. magnifica were cut off under a microscope, and the two ends of the antennae were connected to the corresponding electrodes using a capillary tube. The 1-day-old and 7-day-old W. magnifica were selected for the experiment. Each sample was repeated three times for male and female, and each antenna was used once.
Gas Chromatography (GC) (Agilent) conditions: the initial temperature was 120 • C, kept for 1 min, and then increased to 280 • C at a rate of 20 • C/min, and kept for 15 min; 5:10 split injection, each injection of 1 µL.
Adult antennae of 5-day-old to 7-day-old W. magnifica were connected to an antenna potentiometer (SYNTECH). We took 10µL of the sample to be tested and dropped it on a 30 mm × 10 mm filter paper and placed it in a clean container. After the solvent was volatilized for 5 min, the antennae were stimulated. N-hexane was used as a blank control, and blank control experiments were conducted for each antenna before and after the test.
The calculation formula of EAG relative reaction value is as follows: EAG relative reaction value = (2 × Test the EAG value of the sample) ÷ (EAG value of solvent control before testing sample + EAG value of solvent control after testing sample).
The response data were analyzed and plotted by Graphpad Prism 8.0.1. One-Way ANOVA was used to analyze the EAG response of the same sex to different test samples and the EAG response of different sexes to the same test sample.
We selected 5-day-old to 7-day-old W. magnifica for behavioral experiments. Only one W. magnifica was placed into a Y-shaped tube each time, and the test time was 5 min. When it entered more than half of the side arm and remained there for at least 30 s, the results were recorded. If no choice was made within 5 min, the behavioral experiment for the W. magnifica was stopped. A group of 20 females and 20 males were selected, and the tests were repeated three times. N-hexane was used as blank control, and the experiment was carried out at room temperature.
Graphpad Prism was used for the t-test analysis of the results and plotted by Origin PRO.

Behavioral Experiment of W. magnifica on the Mixture of Methylheptenone and 1-octen-3-ol in Different Proportions
Two compounds were prepared according to the following table (Table 3), and all reagents were prepared 1 h before the experiment. Table 3. The proportion of the mixture.

Reagent Mixing Ratio
Methylheptenone:1-octen-3-ol 1:1 1:0.5 0.5:1 We selected 5-day-old to 7-day-old W. magnifica for behavioral experiments. Only one W. magnifica was placed into a Y-shaped tube each time, and the test time was 5 min. When it entered more than half of the side arm and remained there for at least 30 s, the results were recorded. If no choice was made within 5 min, the behavior experiment for the W. magnifica was stopped. A group of 20 females and 20 males were selected, and the tests were repeated three times. Distilled water was used as blank control, and the experiment was carried out at room temperature.
Our previous studies found that the behavioral response of W. magnifica mainly rely on olfactory sensors, so we did not apply light-avoidance treatment in this experiment. We took 2 mL of prepared reagent and put it into the bait basin of a fly catcher. Different concentrations of single products were compared with the same amount of n-hexane, while different proportions of mixtures were compared with the same amount of distilled water. The fly catcher was randomly hung in a fly cage. In this experiment, a hundred males and a hundred females were selected each time, and the experiment was repeated three times. Each experiment lasted for 12 h, and the data were recorded every 2 h.
The experiment data were analyzed and plotted by Graphpad Prism.  (Figures 1-3). The results showed that the second group detected more abundant sample information.

Liquid Mass
one W. magnifica was placed into a Y-shaped tube each time, and the test time was 5 min. When it entered more than half of the side arm and remained there for at least 30 s, the results were recorded. If no choice was made within 5 min, the behavior experiment for the W. magnifica was stopped. A group of 20 females and 20 males were selected, and the tests were repeated three times. Distilled water was used as blank control, and the experiment was carried out at room temperature.
Our previous studies found that the behavioral response of W. magnifica mainly rely on olfactory sensors, so we did not apply light-avoidance treatment in this experiment. We took 2 mL of prepared reagent and put it into the bait basin of a fly catcher. Different concentrations of single products were compared with the same amount of n-hexane, while different proportions of mixtures were compared with the same amount of distilled water. The fly catcher was randomly hung in a fly cage. In this experiment, a hundred males and a hundred females were selected each time, and the experiment was repeated three times. Each experiment lasted for 12 h, and the data were recorded every 2 h.
The experiment data were analyzed and plotted by Graphpad Prism.

UPLC-QTof/MS BPI Total Ion Chromatogram
UPLC-QTof/MS BPI total ion chromatograms of the three groups of samples were respectively drawn by ACQUITY UPLC H-Class Ultra Performance Liquid Chromatograph (Figures 1-3). The results showed that the second group detected more abundant sample information.

Structure Determination of the Main Components
The raw data were collected by ACQUITY UPLC H-Class Ultra Performance Liquid Chromatograph; the UNIFI was used for the qualitative analysis. Setting appropriate ac quisition parameters and using the overall workflow of UNIFI software were combined with a self-built database of common vaginal compounds for data processing and struc ture matching. A total of 140 vaginal compounds of Bactrian camels were identified b the software, among which, 9 compounds with a high matching degree were identified ( Figure 4).

Structure Determination of the Main Components
The raw data were collected by ACQUITY UPLC H-Class Ultra Performance Liquid Chromatograph; the UNIFI was used for the qualitative analysis. Setting appropriate acquisition parameters and using the overall workflow of UNIFI software were combined with a self-built database of common vaginal compounds for data processing and structure matching. A total of 140 vaginal compounds of Bactrian camels were identified by the software, among which, 9 compounds with a high matching degree were identified ( Figure 4).

Structure Determination of the Main Components
The raw data were collected by ACQUITY UPLC H-Class Ultra Performance Liqui Chromatograph; the UNIFI was used for the qualitative analysis. Setting appropriate ac quisition parameters and using the overall workflow of UNIFI software were combine with a self-built database of common vaginal compounds for data processing and struc ture matching. A total of 140 vaginal compounds of Bactrian camels were identified b the software, among which, 9 compounds with a high matching degree were identifie ( Figure 4).

Multivariate Statistical Analysis-PCA Analysis
A PCA analysis was performed on the test data and a score plot was drawn ( Figure 5). The PCA intuitively shows the separation of different groups of samples, that is, the obvious separation trend of groups of samples.

Multivariate Statistical Analysis-PCA Analysis
A PCA analysis was performed on the test data and a score plot was drawn ( Figure  5). The PCA intuitively shows the separation of different groups of samples, that is, the obvious separation trend of groups of samples.

Analysis Results of OPLS-DA
An OPLS discriminant analysis was performed on the test data of each of two groups of samples and an OPLS-DAS plot ("S" shape) was drawn to show the difference between the two groups. The compounds with the most significant difference between the two groups of samples were located on both sides of the "S" shape, which were potentia markers with high research value [29].
There are significant differences in the score plots among all groups (Figures 6-8) and the S-plot among all groups is shown in Figures 9-11 (the green part is the same part the blue and red parts at both ends are significant differences). The results showed tha the contents of methylheptenone and 1-octen-3-ol were significantly increased in group 2 compared with group 1 and group 3, while propyl butyrate was significantly increased in group 3 compared with the other two groups.

Analysis Results of OPLS-DA
An OPLS discriminant analysis was performed on the test data of each of two groups of samples and an OPLS-DAS plot ("S" shape) was drawn to show the difference between the two groups. The compounds with the most significant difference between the two groups of samples were located on both sides of the "S" shape, which were potential markers with high research value [29].
There are significant differences in the score plots among all groups ( Figures 6-8), and the S-plot among all groups is shown in Figures 9-11 (the green part is the same part; the blue and red parts at both ends are significant differences). The results showed that the contents of methylheptenone and 1-octen-3-ol were significantly increased in group 2 compared with group 1 and group 3, while propyl butyrate was significantly increased in group 3 compared with the other two groups. A PCA analysis was performed on the test data and a score plot was drawn ( Figure  5). The PCA intuitively shows the separation of different groups of samples, that is, the obvious separation trend of groups of samples.

Analysis Results of OPLS-DA
An OPLS discriminant analysis was performed on the test data of each of two groups of samples and an OPLS-DAS plot ("S" shape) was drawn to show the difference between the two groups. The compounds with the most significant difference between the two groups of samples were located on both sides of the "S" shape, which were potential markers with high research value [29].
There are significant differences in the score plots among all groups ( Figures 6-8), and the S-plot among all groups is shown in Figures 9-11 (the green part is the same part; the blue and red parts at both ends are significant differences). The results showed that the contents of methylheptenone and 1-octen-3-ol were significantly increased in group 2 compared with group 1 and group 3, while propyl butyrate was significantly increased in group 3 compared with the other two groups.

GC-EAD Results of Methylheptenone, 1-octen-3-ol, and Propyl Butyrate
The results of GC-EAD showed that female and male W. magnifica at different ages responded to the three compounds ( Figures 12-17).

GC-EAD Results of Methylheptenone, 1-octen-3-ol, and Propyl Butyrate
The results of GC-EAD showed that female and male W. magnifica at different ages responded to the three compounds ( Figures 12-17).

GC-EAD Results of Methylheptenone, 1-octen-3-ol, and Propyl Butyrate
The results of GC-EAD showed that female and male W. magnifica at different ages responded to the three compounds ( Figures 12-17).

GC-EAD Results of Methylheptenone, 1-octen-3-ol, and Propyl Butyrate
The results of GC-EAD showed that female and male W. magnifica at different ages responded to the three compounds ( Figures 12-17).

EAG Results of Three Compounds in Different Concentrations for Male W. magnifica
There was no significant difference in the EAG relative response value of male W. magnifica to different concentrations of methylheptenone, 1-octen-3-ol, and propyl butyrate (p > 0.05) (    There was no significant difference in the EAG relative response value of male W. magnifica to different concentrations of methylheptenone, 1-octen-3-ol, and propyl butyrate (p > 0.05) (   There was no significant difference in the EAG relative response value of m magnifica to different concentrations of methylheptenone, 1-octen-3-ol, and propyl ate (p > 0.05) (

EAG Results of three Compounds in Different Concentrations for Female W. magnifica
There was no significant difference in the EAG relative response values of female W. magnifica to different concentrations of 1-octen-3-ol and propyl butyrate (p > 0.05). The EAG relative response values of 1μg/μL methylheptenone were significantly higher than those of 10 −4 μg/μL and 10 −3 μg/μL (0.01 < p < 0.05), but the other concentrations were not significant (p > 0.05) (

EAG Results of three Compounds in Different Concentrations for Female W. magnifica
There was no significant difference in the EAG relative response values of female W. magnifica to different concentrations of 1-octen-3-ol and propyl butyrate (p > 0.05). The EAG relative response values of 1μg/μL methylheptenone were significantly higher than those of 10 −4 μg/μL and 10 −3 μg/μL (0.01 < p < 0.05), but the other concentrations were not significant (p > 0.05) ( Table 5, Figures 21-23).

EAG Results of three Compounds in Different Concentrations for Female W. magnifica
There was no significant difference in the EAG relative response values of female W. magnifica to different concentrations of 1-octen-3-ol and propyl butyrate (p > 0.05). The EAG relative response values of 1µg/µL methylheptenone were significantly higher than those of 10 −4 µg/µL and 10 −3 µg/µL (0.01 < p < 0.05), but the other concentrations were not significant (p > 0.05) ( Table 5, Figures 21-23).

EAG Results of three Compounds in Different Concentrations for Female W. magnifica
There was no significant difference in the EAG relative response values of female W. magnifica to different concentrations of 1-octen-3-ol and propyl butyrate (p > 0.05). The EAG relative response values of 1μg/μL methylheptenone were significantly higher than those of 10 −4 μg/μL and 10 −3 μg/μL (0.01 < p < 0.05), but the other concentrations were not significant (p > 0.05) ( Table 5, Figures 21-23).    The results showed that there was no significant difference in the EAG relative reaction value of different sexes to methylheptenone, 1-octen-3-ol and propyl butyrate (p > 0.05) (Figures 24-26).

The Study of Comparative on EAG Responses of Different sexes of W. magnifica to the Same Compound
The results showed that there was no significant difference in the EAG relative reaction value of different sexes to methylheptenone, 1-octen-3-ol and propyl butyrate (p > 0.05) (Figures 24-26).

The Study of Comparative on EAG Responses of Different sexes of W. magnifica to the Same Compound
The results showed that there was no significant difference in the EAG relative reaction value of different sexes to methylheptenone, 1-octen-3-ol and propyl butyrate (p > 0.05) (Figures 24-26).

The Study of Comparative on EAG Responses of Different sexes of W. magnifica to the Same Compound
The results showed that there was no significant difference in the EAG relative reaction value of different sexes to methylheptenone, 1-octen-3-ol and propyl butyrate (p > 0.05) (Figures 24-26).

The Study of Comparative on EAG Responses of Different sexes of W. magnifica to the Same Compound
The results showed that there was no significant difference in the EAG relative reaction value of different sexes to methylheptenone, 1-octen-3-ol and propyl butyrate (p > 0.05) (Figures 24-26).

Discussion
At present, the treatment of vaginal myiasis of Bactrian camels rely on pesticide deworming, but this method can't eradicate the disease, and would be cause environmental pollution. The results of liquid mass spectrometry showed that methylheptenone, 1-octen-

Discussion
At present, the treatment of vaginal myiasis of Bactrian camels rely on pesticide deworming, but this method can't eradicate the disease, and would be cause environmental pollution. The results of liquid mass spectrometry showed that methylheptenone, 1-octen-3-ol, and propyl butyrate were the important components with attractive efficacy to W. magnifica in the vaginal secretions of Bactrian camels.
The GC-EAD and EAG results of W. magnifica to methylheptenone, 1-octen-3-ol, and propyl butyrate showed that there was a response to the three compounds at different ages of W. magnifical. Behavioral experiments and trapping experiments were carried out on W. magnifica with different concentrations of methylheptenone, 1-octen-3-ol, propyl butyrate, and mixtures of methylheptenone and 1-octen-3-ol in different proportions, the results showed that, except for propyl butyrate, the higher the concentration of the other two compounds, the more attractive to the W. magnifica, and the 1:1 ratio mixture of these could enhance the attraction to W. magnifical.
A host is indispensable for insect survival. Host volatiles play an important role in mate selection and reproduction for insects [30]. For example, the volatile of cow urine is attractive to Musca autumnalis [31]. Studies showed that lepidopteron insects rely on smell and plant volatiles for host selection, and they can identify and locate their hosts by sensing specific compounds from their hosts [32]. At present, GC-EAD and EAG are mainly used in the research of insects and their host plants and non-host plants.
The main function of GC-EAD is to detect whether there are substances that have relevant reactions to insect antennae from the crude extracts of host volatiles, and then using the separation and discrimination ability of GC to screen out the corresponding substances. EAG mainly reflects the reactions of insects to different compounds, so as to screen out the substances with stronger reactions. The results showed that the three compounds had EAG responses to both male and female W. magnifica, and the response of females was higher than males in general. Studies have found that ®-3-hydroxy-2-hexanone had EAG responses to both male and female Diploschema rotundicolle [33], similar to our results, while other studies have shown that the EAG response of male moths to (Z)-3-hexenol butyrate, (Z)-3-hexene-1-ol, and 3,7-dimethyl-1,3,7-octantriene was significantly higher than that of female moths [34]. The insect species, sex, sensitivity to compounds, and the concentration of compounds affects the EAG response. For example, compared with Apis mellifera, the EAG response of Apis cerana to queen mandibular pheromone is always weaker [35]; 1 µg of compound can cause a higher EAG response of Triatoma dimidiate complex [36]. In addition, the response of antennae to volatiles varies with the time of day because the olfactory response and the pulse-tracking ability of antennae follow the circadian rhythm of insects [37].
In addition to the use of pesticides, using insect pheromones and volatile compounds related to food to trap insect pests has become an effective pest control method. At present, there is no report on biological control methods of W. magnifica, but some studies have found that using human-odor extracts to attract adult female mosquitoes into traps can reduce the survival rate of mosquitoes [38]. Kaidi Cui found that 1-octen-3-ol is attractive to blood-sucking insects, and can be used as an attractant of mosquitoes [39]; in addition, it can inhibit Drosophila melanogaster and Rhynchophorus ferrugineus from ovipositing [40,41]. Many studies have found that mixing host volatiles can change the trapping rate [42]. For example, compared with 1-octen-3-ol alone, mixed use of it with CO 2 can improve its trapping rate [43,44]; vanillin and DEET used at the same time can enhance the repellency of DEET to mosquitoes [45]; and a synthetic terpenoid mixture has strong attraction to houseflies [46]. Thus, adding particular combinations of attractants to traps can improve the trapping rate of insects [47]. Many insects detect their specific proportion of key volatiles to find their hosts; therefore, even a few small changes in proportions may increase or decrease the attraction of hosts to insects [48].

Conclusions
Our study showed that methylheptenone, 1-octen-3-ol, and propyl butyrate can cause an antennae reaction of W. magnifica. Different concentrations of methylheptenone, 1-octen-3-ol, and propyl butyrate are attractive to W. magnifica, and the mixture of methylheptenone and 1-octen-3-ol at the ratio of 1:1 can attract more W. magnifica. This experiment laid a foundation for biological control of vaginal myiasis in Bactrian camels.

Institutional Review Board Statement:
The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of Inner Mongolia Agricultural University (No. NND2021016), and strictly followed animal welfare and ethical guidelines.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The datasets during and/or analyzed study available from the corresponding author and can be provided upon request.