Introduction

Demodex mites, specifically Demodex folliculorum and Demodex brevis, are parasitic organisms that reside on the human body and have significant implications for ocular health1,2. Demodex folliculorum primarily infests the roots of eyelashes and hair follicles3,4, feeding on the epithelial cells that surround these structures, potentially leading to trichiasis and eyelash loss5; In contrast, Demodex brevis is more commonly found within the meibomian glands, where it can obstruc glandular orifices, impede secretion, and result in meibomian gland dysfunction6. Consequently, the prevalence of Demodex folliculorum is significantly higher than that of Demodex brevis when examining the eyelashes of the general population or patients with blepharitis7. Morphologically, adult Demodex mites exhibit a worm-like appearance with a milky white, translucent body, which is segmented into the gnathosoma, podosoma, and opisthosoma. The gnathosoma is trapezoidal, the podosoma features four pairs of segmented, sleeve-like legs, and the opisthosoma is finger-shaped with distinct ring-like markings. The life cycle of Demodex mites encompasses five stages: egg, larva, protonymph, tritonymph, and adult, with the developmental period from egg to larval estimated at 14–18 days, followed by a 5-day adult stage. Female mites can survive for an additional 5 days post-reproduction8.

The prevalence of Demodex mites is notably high, with infection rates reported to vary from 27 to 100%, peaking at an alarming 97.86% in certain regions of China. This rate demonstrates a strong correlation with age, with 84% of individuals above 60 years being affected, and a striking 100% in those over 70 years old9. Despite the high prevalence, current clinical practice is yet to establish universally effective treatment methods for ocular conditions associated with Demodex infestations. Interventions such as Intense Pulsed Light (IPL), Optimized Pulse Technology (OPT), meibomian gland expression, and the use of mites patches require further investigation to confirm their efficacy. Tea tree oil (TTO) stands out as a potential acaricide, yet its diverse product formulations, varied application techniques, and the need for safety validation present ongoing challenges for researchers. The search for efficacious acaricidal agents remains an active area of clinical exploration. This study delves into the survival capabilities and morphological responses of Demodex mites under a range of temperatures and culture media. Beyond TTO, we have examined several standard culture media, including Phosphate-Buffered Saline (PBS)10, deionized water, physiological saline11, Propidium Iodide (PI) staining solution10,12, liquid paraffin, glycerol12, and a blank medium11. Qur goal is to identify optimal in vitro culture conditions that support the development of Demodex mites, thereby establishing a foundational experimental framework for the screening of potential clinical therapeutics.

Results

Activity change

Utilizing a light microscope, we assessed the vitality of Demodex mites by monitoring the movement of their claws, quantifying their activity on a scale of “+ to +++” based on the degree of motion observed in the claw limbs13.

Employing this grading system, we determined that at room temperature, the survival span of Demodex mites in all tested culture media did not exceed one week. Notably, mites in liquid paraffin exhibited the most extended survival, enduring for up to 5 days, whereas those in pure water, glycerol, and blank medium had the shortest survival span of only 2 days (Table 1). At a reduced temperature of 4 °C, the longevity of Demodex mites in liquid paraffin was significantly increased, reaching a maximum of 12 days. The majority of mites survived for less than a week; however, a small subset, approximately 19%, retained claw movement even after one week. The survival times in various media at 4 °C were as follows: 5 µg/ml propidium iodide (PI), PBS, and blank medium allowed survival of 8 days; pure water and glycerol sustained mites for 5 days; 0.9% physiological saline supported survival for 4 days; and 30% tea tree oil (TTO) resulted in the shortest survival time of less than 1 day. We observed a rapid loss of mobility in Demodex mites within 5 to 10 min post-exposure to 30% TTO, indicating its potent acaricidal properties (Table 2).

Table 1 Survival time of Demodex folliculorum in the eight groups at 16–22℃.
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Table 2 Survival time of Demodex folliculorum in the eight groups at 4℃.
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Morphological changes

Using Phosphate-Buffered Saline (PBS), liquid paraffin and 30% tea tree oil (TTO) as representative media, we conducted morphological assessments of Demodex mites under the light microscope. Initially, the joint ganglia of the mites were observed to be dark brown. Over time and with the decline in their motility, the color, size, and shape of the mites in both liquid paraffin and PBS at 4 ℃ remained largely unchanged. However, in the PBS group, the joint ganglia exhibited a gradual lightening in color (Fig. 1A-D). In contrast, the joint ganglia in liquid paraffin showed a marginally reduced size with no significant color alteration (Fig. 2E-H). Despite the fluid nature of the culture medium, the position of the Demodex mites continued to shift even after their motility ceased.

At room temperature, a noticeable change in the Demodex mites’ appearance was observed in PBS: their bodies became significantly lighter in color. Post-mortem, the bodies became transparent, with an observable increase in internal bubbles. The volume of the Demodex mites expanded following the loss of motility, resulting in a larger body size compared to their initial state (Fig. 3I-L). The body color of Demodex mites gradually becomes lighter in liquid paraffin, and compared to PBS, the dead volume of Demodex mites in liquid paraffin did not show any expansion (Fig. 4M-P).

Upon the introduction of 30% tea tree oil (TTO), the Demodex mites exhibited an immediate and violent contraction and twisting of their bodies. Notably, the junction between the foot body and terminal body constricted, becoming noticeably thinner (Fig. 5Q-T). The color of Demodex mites’ bodies faded to a pale hue, and the joint ganglia became so diminished that the mites were often indistinguishable. As the drug’s effect persisted, the motility of the Demodex mites’ feet gradually diminished, culminating in the relaxation and death of the organisms. In certain samples, the contracted state was still be observable 24 h post-administration, with the recovery of the contracted area typically occurreing within a range of 20 mintures to 2 days. However, in some instances, the Demodex mites failed to exhibit the characteristic shrinkage following TTO exposure.

Fig. 1
figure 1

Morphological comparison of Demodex samples in PBS at 4℃. A, Demodex before adding PBS; B, There was no obvious Demodex mites’ morphological change after adding PBS; C, Demodex samples added to PBS for 7 days, the joint ganglia gradually disappeared and spread over time. D, After adding PBS for 14 days, the morphology of Demodex mites showed no significant change compared to 7 days.

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Fig. 2
figure 2

Morphological comparison of Demodex samples in liquid paraffin at 4℃. E, Demodex before adding liquid paraffin; F, There was no significant change in the morphology change after adding liquid paraffin; G, After adding liquid paraffin for 7 days, there was no significant change in the morphology of Demodex mites compared to just adding liquid paraffin. H, After being added to liquid paraffin for 14 days, the joint ganglia range of Demodex mites decreased, but the color remained unchanged.

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Fig. 3
figure 3

Morphological comparison of Demodex samples in PBS at 16–22℃. I, Demodex before adding PBS; J, There was no significant change in the morphology change after adding PBS; K, After adding to PBS for 2 days, the joint ganglion gradually disappeared over time. L, After adding PBS for 5 days, the color of the Demodex body becomes significantly lighter, and a large number of bubbles can be seen inside the body.

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Fig. 4
figure 4

Morphological comparison of Demodex samples in liquid paraffin at 16–22℃. M, Demodex before adding liquid paraffin; N, There was no obvious Demodex morphological change after adding liquid paraffin; O, After adding to liquid paraffin for 2 days, the joint ganglia range of Demodex mites decreased and the color became lighter. P, After adding liquid paraffin for 5 days, the body volume of Demodex mites slightly decreased, while the joint ganglia continued to shrink and become shallower.

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Fig. 5
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Morphological comparison of Demodex samples in TTO. Q, The Demodex before adding the TTO; R, The Demodex body became shorter and contracted after adding the TTO for 5 min; S, The colour of the Demodex body gradually became weak to transparent over time; T, With the weakening of drug action, the contracted part of the worm recovered and the colour became darker.

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Observation on the eggs and larvae of demodex

In this study, we extended our investigation to include the eggs and larvae of Demodex mites, marking the first time their vitality has been tracked and observed. We noted that the eggs and larvae lacked observable movement in their bodies or claws. Additionally, they are characterized by a lighter and more translucent appearance compared to adult mites, making them more challenging to discern. Our research revealed that both the eggs and larvae of Demodex mites display auto-fluorescence, however, this property in the eggs is transient, persisting for only up to 24 h (Fig. 6). The larvae, which have begun to develop joint ganglia, exhibit a weak auto-fluorescence that essentially fades away after a period of 5 days ( Fig. 7).

Fig. 6
figure 6

egg.A, Morphology of Demodex egg after sampling; egg.B, Blue auto-fluorescence of Demodex egg after sampling; egg.C, PI staining fluorescence of Demodex egg after sampling; egg.a, Morphology of Demodex egg 1 day after sampling; egg.b, Blue auto-fluorescence of Demodex egg 1 day after sampling; egg.c, PI staining fluorescence of Demodex egg 1 day after sampling.

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Fig. 7
figure 7

larva.A, Morphology of Demodex larvae after sampling larva.B, Blue autofluorescence of Demodex larvae after sampling larva.C, PI staining fluorescence of Demodex larvae after sampling larva.a, Morphology of Demodex larvae 5 days after sampling; larva.b, Blue autofluorescence of Demodex larvae 5 days after sampling; larva.c, PI staining fluorescence of Demodex larvae 5 days after sampling.

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Discussion

Previous studies have indicated that the in vitro survival of Demodex mites is influenced by factors such as temperature, humidity, culture medium, and pH value. Our research substantiates the significance of temperature and medium in the in vitro culture of Demodex mites, aligning with certain existing findings11,13,14. In our experiments, the average survival time for Demodex mites in various culture media at a temperature range of 16–22 °C was observed to be 3 days, which increased to 6.86 days at 4 °C, excluding 30% tea tree oil (TTO) due to its rapid lethal effect on the mites. This suggests that a lower temperature of 4 °C is more effective for sustaining the in vitro survival of Demodex mites.

Our study particularly examines the morphological alterations of Demodex mites in different culture media at two distinct temperatures, with a focus on Phosphate-Buffered Saline (PBS) and liquid paraffin. Upon the addition of 30% TTO, we observed violent contractions and deformations in the mites under high magnification. Following an initial period of agitation, the mites entered a state of inhibition, gradually relaxing, recovering, slowing in movement, and ultimately succumbing to death. Throughout this process, the integrity of the mite’s body wall was maintained, with no observed ruptures. Apart from the effects of 30% TTO, we noted nuanced differences in the morphological changes across other culture media. As the mites progressively died, their bodies became lighter or increasingly transparent, and their joint ganglia became less distinct and more diffuse. Compared to the conditions at 16–22 °C, these morphological changes were observed to be slower at 4 °C. It is important to acknowledge the limitations of our study, such as a small sample size, which may introduce some degree of experimental variability.

Propidium iodide (PI) serves as a reliable indicator of cell viability, leveraging its exclusion by living cells. The entry of PI into a cell is contingent upon membrane permeability; it does not stain live or early apoptotic cells due to the integrity of the plasma membrane. In contrast, during late apoptosis and necrosis, the compromised integrity of plasma and nuclear membranes permits PI to cross, intercalate with nucleic acids, and emit red fluorescence15. Our findings reveal that, despite the inactivity of Demodex mite eggs and larvae, PI fails to impart a red coloration when they exhibit blue auto-fluorescence. This suggests that inactive Demodex mites may still be in a state of survival, underscoring the need for further research to ascertain whether auto-fluorescence can be an indicator of mite vitality.

Currently, no specific drug is available for the clinical treatment of ocular Demodex infections. In recent years, extensive research has focused on the ecology and acaricidal potential against Demodex mites, with in vitro experiments conducted on crude extracts of various natural plants yielding promising results. The in vitro culture of Demodex mites is instrumental for the screening and assessment of potential clinical therapeutics, which is also the objective of our study.

Materials and methods

Materials

30% TTO, PBS, pure water, 0.9% physiological saline, 5 µg/ml PI, liquid paraffin, glycerol, ophthalmic forceps, wet box, glass slide, cover glass slide.

Methods

Sample source

This study was conducted in accordance with the principles of the Helsinki Declaration and obtained approval from the Institutional Review Committee of the Affiliated Traditional Chinese Medicine Hospital of Nanjing University of Chinese Medicine. Written informed consent was obtained from all participants, who subsequently underwent comprehensive eye examinations and external photography as part of the routine procedure.Samples were collected by extracting two eyelashes from each of the nasal, central, and temporal regions of the upper and lower eyelids of the enrolled patients. This process utilized inverted eyelash forceps, ensuring six eyelashes were collected per eye. The collected eyelashes were carefully placed on a microscope slide to prevent the escape of any Demodex mites. Immediately following the removal of the specimen, the culture medium was applied to the sample, which was then covered with a glass slide. The specimens were subsequently examined under a microscope to assess the presence and characteristics of the mites.

Experimen design

The study employed a factorial design, investigating the effects of two independent variables: temperature and culture medium. The temperature was manipulated at two levels: ambient room temperature ranging from 16 to 22 °C and a controlled low temperature of 4 °C. The culture media were categorized into eight distinct types: 30% tea tree oil (TTO), Phosphate-Buffered Saline (PBS), deionized water, 0.9% physiological saline, 5 µg/ml solution of propidium iodide (PI), liquid paraffin, glycerol, and a blank medium devoid of any additives.

Test grouping

To each slide, 60–70 µl of the respective culture medium was meticulously added and spread to ensure uniform contact with the body of Demodex mites. The slides were then placed in incubators set to the two aforementioned temperature levels, maintaining a relative humidity of 85–90%. The samples were regularly removed for microscopic examination to assess the survival characteristics of the Demodex mites.

Activity identification

The assessment of mite activity was based on the following standardized criteria:

  • “−”(Negative): A mite was considered inactive if its chelicera or legs remained motionless for a continuous period of 1 minute, and this inactivity persisted after a subsequent observation made 30 minutes later, indicating the mite was deceased.

  • “±”(Indeterminate): A mite was classified as indeterminate if it exhibited no movement after being maintained at a low temperature for 4 hours but resumed movement upon recovery (anabiosis), suggesting a state of suspended animation.

  • “+”(Weak Movement): A mite displayed weak movement, characterized by the motion of its chelicera or one to two legs, with movements occurring one to two times per minute.

  • “++”(Moderate Movement): A mite exhibited moderate activity, with its chelicera or three to five legs moving with noticeable frequency, three to five times per minute.

  • “+++”(Strong Movement): A mite demonstrated strong activity, with the motion of its chelicera or all six to eight legs, showing active movement more than six times per minute.