Portable constant-temperature piercing-sucking type insect feeding system and method

Abstract

The invention relates to a portable constant-temperature piercing-sucking type insect feeding system and method, and belongs to the technical field of insect experiment equipment. In order to mainly solve the problems that a traditional mosquito feeding device is insufficient in temperature control precision, the temperature of a permeable membrane area is obviously interfered by the environment and the like, closed-loop temperature control is achieved by arranging a circular mounting hole with an internal thread at the bottom of a heating and heat preservation container and integrating a miniature thermistor and a PID temperature controller on the inner wall; external threads are arranged outside the connecting sleeve and are screwed and fixed with the mounting hole; the inner diameter of the connecting sleeve and the outer diameter of the lower part of the blood container form an interference fit structure; after the blood container is sleeved with the connecting sleeve, the bottom of the blood container and the permeable membrane extend downwards to form an exposed area. The system is mainly used for constant-temperature feeding of mosquitoes in a laboratory or a field environment, and the temperature stability and the operation reliability are remarkably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of insect experimental equipment, and in particular relates to a portable constant-temperature piercing-sucking insect feeding system and method. Background Art

[0002] Maintaining a stable feeding environment for mosquitoes is crucial in piercing-sucking entomology research, especially in experiments related to mosquito-borne diseases. Current mosquito feeding devices commonly used in laboratories typically utilize a constant-temperature water bath with an open blood container (such as a Petri dish or beaker) covered with a permeable membrane to simulate the surface of biological skin. However, this technical solution has the following significant drawbacks: 1. Insufficient temperature control accuracy. Existing devices mostly rely on simple thermostats or open-loop heating systems, and the temperature fluctuation range often exceeds ±1°C. This defect stems from two aspects: first, the water bath device still has a thermal hysteresis effect after reaching the set temperature, causing the actual temperature to continue to rise by 0.5-1.2°C; second, the open container is exposed to the air, and changes in environmental airflow such as air conditioning and people walking around exacerbate the temperature fluctuations on the surface of the permeable membrane. Because mosquitoes are extremely sensitive to feeding temperature, excessive temperature fluctuations will affect their feeding. When trying to use a higher-precision thermostat, the device size increases and energy consumption increases significantly, making it difficult to meet portability requirements.

[0003] 2. Poor blood container stability. In traditional devices, blood containers are placed directly on a waterbath float or suspension structure, lacking an effective securing mechanism. The impact of mosquitoes landing to feed can easily cause the container to shift or even tip over, disrupting the experiment. The non-rigid contact between the bottom of the container and the water bath liquid surface affects heat transfer efficiency due to fluctuations in the water flow. Attempts have been made to secure the blood container with additional weights or clips, but these increase the device's size and make it difficult to accommodate containers of varying sizes.

[0004] 3. Deterioration of the temperature environment of the permeable membrane. The permeable membrane is the direct interface for mosquitoes to bite and suck food, and its temperature uniformity directly affects the feeding efficiency. However, in the existing technology, when the membrane surface is completely exposed to the air, the edge area dissipates heat due to convection, resulting in a temperature gradient of more than 1°C. When the center is 38°C, the edge is only around 35.5-37°C, forming a low-temperature zone that affects mosquito feeding. Although immersing the lower half of the container in a water bath improves temperature uniformity, it increases the risk of membrane rupture and the probability of blood contamination. Optimizing the temperature distribution on the membrane surface requires balancing heat conduction efficiency and physical isolation, but the space limitations of miniaturized devices such as micro heat pipes make it difficult to integrate thermal compensation structures.

[0005] These defects lead to temperature fluctuations that exacerbate the risk of container displacement, while container instability further degrades the membrane surface temperature. Within the constraints of portability, simultaneously addressing these three issues presents the following challenges: high-precision temperature control components such as multiple sensors require a large footprint; strengthening container fixation requires additional mechanical components, which obstructs the heat transfer path; and reducing temperature fluctuations requires continuous active temperature control, which conflicts with the low power consumption requirements of battery-powered devices. Therefore, a new feeding system design that balances temperature stability, mechanical reliability, and portability is urgently needed. Summary of the Invention

[0006] The present invention addresses the following technical problems: The constant temperature control accuracy in traditional mosquito feeding devices is insufficient, with fluctuations usually greater than ±1°C. The blood container lacks a stable fixing mechanism, making it easy to move and overturn. The temperature in the permeable membrane area is significantly affected by environmental disturbances, making it difficult to maintain a stable feeding microenvironment.

[0007] The flat-mounted blood container has low heat transfer efficiency, the air temperature convection around the permeable membrane causes increased fluctuations, and the lack of a physical support surface for mosquitoes when feeding reduces the success rate.

[0008] The edge of the permeable membrane forms a temperature gradient due to the growth of the heat conduction path, the mechanical positioning error causes uneven membrane tension, and traditional rigid connections have the risk of liquid leakage.

[0009] The residual volume of straight blood containers is large, usually greater than 15%. The membrane edge forms an ineffective feeding area due to the meniscus effect, and the contact angle between blood and the container wall is too large, causing capillary blockage.

[0010] Thin permeable membranes are easily deformed and damaged under blood pressure, and the mismatch in thermal expansion coefficients of traditional support meshes leads to high-temperature fitting gaps.

[0011] The traditional feeding method causes the mosquitoes to respond to stress due to the rapid heating rate. The temperature difference on the membrane surface leads to a decrease in feeding efficiency. When the liquid level drops, there is no rehydration mechanism, resulting in waste.

[0012] Thermal inertia during the single-step heating process causes temperature overshoot, the fixed flow rate cannot compensate for the temperature difference on the membrane surface in real time, the blood viscosity is not optimized, affecting fluidity, and the residual cleaning is not thorough.

[0013] The present invention provides a portable constant temperature piercing-sucking insect feeding system, comprising: The heating and insulation container has at least one circular mounting hole with an internal thread on its bottom, and a micro thermistor is provided on the inner wall of the heating and insulation container and is connected to a PID temperature controller for accurately controlling the temperature of the interior space of the container; a blood container having a bottom opening downward; a permeable membrane, sealingly covering the open bottom of the blood container; A connecting sleeve, the outer surface of which is provided with an external thread matching the internal thread of the circular mounting hole; The inner diameter of the connecting sleeve forms an interference fit structure with the outer diameter of the lower part of the blood container; the connecting sleeve is screwed into and fastened to the circular mounting hole at the bottom of the heating and insulation container through an external thread; The blood container is inserted downwardly and fixed in the connecting sleeve, and a tight connection and positioning are achieved through an interference fit structure. At the same time, the bottom of the blood container and the permeable membrane covering it extend downward and are slightly concave, flat, or protruding from the lower end surface of the connecting sleeve, forming an exposed area at the bottom of the heating and insulation container.

[0014] Preferably, the bottom of the heating and insulation container is recessed inward around the position of the circular mounting hole to form a concave area with a coaxial center; the depth (H) of the recessed area is 0.5-2 cm; the side walls and bottom walls of the recessed area are directly surrounded by the insulation liquid inside the heating and insulation container, so that the recessed area forms an outwardly open mosquito feeding space with a relatively stable temperature.

[0015] Preferably, the lower end surface of the connecting sleeve extends in the inner diameter direction to form an annular retaining edge, which is used to abut against the shoulder of the lower outer wall of the blood container when the blood container is inserted downward, thereby limiting the blood container from protruding downward excessively and ensuring the preset exposure height of the permeable membrane; Inside the pipe wall of the connecting sleeve, a thin heat conducting pipe is arranged around the circumference thereof, and the thin heat conducting pipe is arranged on the pipe wall of the connecting sleeve and in the annular baffle area to form a spiral converging flow channel; The upper end of the thin heat-conducting tube is connected to the outlet of the micro-circulation pump through a flexible insulation pipeline, and the inlet of the micro-circulation pump extends to the insulation liquid inside the heating and insulation container through another flexible insulation pipeline. The micro-circulation pump is located outside the heating and insulation container.

[0016] Preferably, the bottom of the blood container is provided with an inverted cone structure, the inverted cone structure gradually increasing in diameter downwards; The inner surface of the inverted cone structure is covered with a bionic hydrophilic coating, the contact angle of the bionic hydrophilic coating is ≤15°, and the coating extends at least 1-2 mm along the inner wall of the blood container to the upper surface edge of the permeable membrane.

[0017] Preferably, an independent cup-shaped support frame is provided at the bottom of the blood container, comprising: The cup-shaped main structure has an inner diameter that forms an interference fit with the outer diameter of the bottom of the blood container, with an interference fit of 0.05-0.1mm and a height that is 80%-90% of the height of the bottom of the blood container; The regular hexagonal grid structure is located at the bottom of the cup-shaped main structure, with a mesh wire diameter of 0.20±0.02mm, an opening side length of 0.8-1.0mm, and an opening ratio ≥65%; A biomimetic hydrophilic coating continuously covers the surface of the grid structure and the inner wall of the cup-shaped main structure, with a coating contact angle of ≤15° and a thickness of 5-10μm; Among them, after the permeable membrane is wrapped around the bottom of the blood container, it is inserted into the cup-shaped support grid as a whole, so that the grid structure fits and supports the entire area of ​​the bottom surface of the permeable membrane.

[0018] The present invention provides a feeding method of a portable constant temperature piercing-sucking insect feeding system, comprising the following steps: Step 1: Screw the external thread of the connecting sleeve into the circular mounting hole at the bottom of the heating and insulation container until the lower end surface of the connecting sleeve contacts the bottom surface of the recessed area; Step 2: Start the PID temperature controller to heat the insulation liquid in the heating and insulation container to a set temperature of 38±0.3°C at a heating rate of 0.5-1°C / min, and maintain this temperature for at least 10 minutes; Step 3: Start the micro-circulation pump and control the flow rate of the insulation liquid through the thin heat pipe and the spiral converging flow channel at 5-10 mL / min. Continue running for at least 5 minutes to stabilize the temperature. Step 4: Cover the bottom of the inverted cone-shaped opening of the blood container with a permeable membrane and seal the edges; insert the blood container vertically downward into the connecting sleeve and secure the blood container in the connecting sleeve by interference fit; and inject blood preheated to 37-39° C. into the blood container; Step 5: Allow the mosquitoes to contact the exposed area of ​​the permeable membrane to feed. When the blood level drops to 1 / 3 of the total height of the container, increase the flow rate to 12-15 mL / min through a micro-circulation pump.

[0019] Preferably, step 2 specifically includes: Step 21, controlling the heating rate to 0.8°C / min until reaching 37.5°C by using a PID temperature controller; Step 22, maintaining a constant temperature of 37.5°C for 5 minutes; Step 23, heating to 38°C at a rate of 0.3°C / min; Step 24: monitor the temperature of the recessed area ≤5 mm away from the installation position of the permeable membrane in real time through a sensor. If the fluctuation value is greater than 0.1° C. within 10 minutes, repeat steps 22 to 23.

[0020] Preferably, step 3 includes the following flow rate optimization control steps: Step 41, initially starting the micro-circulation pump to 5 mL / min; Step 42, obtaining the real-time temperature difference ΔT between the center point and the edge point of the permeable membrane through a sensor; Step 43: If ΔT>0.05°C, the pump speed of the micro-circulation pump is dynamically adjusted according to the formula Vpump=7+25×ΔT (mL / min), and the maximum speed does not exceed 15 mL / min.

[0021] Preferably, in steps 4 and 5: Preheat the blood to 38 ± 0.5°C and maintain the temperature for 10 minutes to reduce the viscosity to 2.8-3.0 mPa·s; When the blood level drops to 1 / 3 of the total height, add 0.5-1 mL of preheated blood from the same batch to the blood container; After a single feeding, the residual blood volume was measured. If it was >0.1 mL, the inner wall of the inverted cone structure was flushed with 42°C saline.

[0022] The present invention has at least the following beneficial effects: 1. By integrating PID temperature control and interference fit structure, the feeding temperature stability and the mechanical stability of the container are significantly improved, forming a controllable exposure area to isolate environmental interference and provide mosquitoes with a uniform and stable feeding microenvironment.

[0023] 2. The recessed area design increases the heat transfer area and reduces edge heat loss. The physical support of the side wall improves the contact stability of the mosquito's feet, thereby increasing the success rate of feeding.

[0024] 3. The annular baffle accurately limits the height of the permeable membrane, the distributed microchannels combined with dynamic circulation eliminate the temperature difference on the membrane surface, and the external magnetic pump design eliminates the risk of leakage, ensuring temperature uniformity and sealing reliability.

[0025] 4. The inverted cone structure cooperates with the super-hydrophilic coating to reduce blood residue, eliminate the ineffective edge area, promote blood spreading to cover the entire surface of the permeable membrane, and increase the effective feeding area.

[0026] 5. The grid support frame matches the thermal expansion coefficient, evenly distributes the pressure on the membrane surface to avoid deformation, and the hydrophilic coating reduces blood adhesion and significantly reduces the membrane damage rate.

[0027] 6. Step-by-step assembly and stepped heating suppress temperature overshoot, dynamic flow rate adjustment compensates for edge heat dissipation, and quantitative fluid replenishment maintains hydraulic stability, comprehensively improving feeding efficiency and blood utilization.

[0028] 7. The insulation section design eliminates system thermal inertia, the temperature difference feedback formula optimizes thermal compensation in real time, blood viscosity control enhances fluidity, and physiological saline flushing thoroughly removes residues to ensure experimental repeatability.

[0029] Other advantages, objectives and features of the present invention will be reflected in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a structural schematic diagram of the heating and heat preservation container of the present invention; Figure 2 This is a schematic structural diagram of the blood container and the connecting sleeve of the present invention; Figure 3 Schematic diagram of the vertical cross-section structure of the portable constant temperature piercing-sucking insect feeding system of the present invention; Figure 4 for Figure 3 A schematic diagram of the first enlarged structure at position A in the middle, where no cup-shaped support grid is set; Figure 5 for Figure 3 A second enlarged diagram of the structure at position A. A cup-shaped support grid is now installed.

[0031] In the figure, 1. Heating and insulation container; 11. Circular mounting hole; 12. Micro thermistor; 13. PID temperature controller; 15. Recessed area; 16. Insulating liquid; 2. Blood container; 21. Shoulder; 22. Inverted cone structure; 23. Cup-shaped support grid; 23a. Cup-shaped main structure; 23b. Regular hexagonal grid structure; 23c. Bionic hydrophilic coating; 3. Permeable membrane; 4. Connecting sleeve; 41. External thread; 42. Annular retaining edge; 43. Thin heat pipe; 44. Spiral converging flow channel; 45. Flexible insulation pipeline; 46. Micro circulation pump; 5. Exposure area. DETAILED DESCRIPTION

[0032] The present invention is further described in detail below with reference to examples so that those skilled in the art can implement the invention with reference to the description.

[0033] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0034] like Figures 1 to 5 As shown, an example of a portable constant temperature piercing-sucking insect feeding system of the present invention includes: The heating and insulation container 1 has at least one circular mounting hole 11 with an internal thread at its bottom, and a micro thermistor 12 is provided on the inner wall of the heating and insulation container 1 and is connected to a PID temperature controller 13 for accurately controlling the temperature of the interior space of the container; A blood container 2 having a bottom opening downward; a permeable membrane 3, sealingly covering the open bottom of the blood container 2; The connecting sleeve 4 has an outer surface provided with an external thread 41 that matches the internal thread of the circular mounting hole 11; The inner diameter of the connecting sleeve 4 forms an interference fit structure with the outer diameter of the lower portion of the blood container 2; the connecting sleeve 4 is screwed into and fastened to the circular mounting hole 11 at the bottom of the heating and heat preservation container 1 through an external thread 41; The blood container 2 is inserted downwardly into and fixed in the connecting sleeve 4, and a tight connection and positioning are achieved through an interference fit structure. At the same time, the bottom of the blood container 2 and the permeable membrane 3 covering it extend downward and are slightly concave, flat, or protruding from the lower end surface of the connecting sleeve 4, forming an exposed area 5 at the bottom of the heating and insulation container 1.

[0035] Specifically, common laboratory mosquito feeding devices in the prior art typically consist of a simple constant-temperature water bath (or hot plate) and a container (such as a beaker or petri dish) containing blood, with the container mouth covered by a permeable membrane (such as Parafilm® or collagen membrane). The water bath provides basic heating, and the blood container is placed directly above the water surface or immersed in the water. Temperature control is relatively rough (e.g., ±1°C or greater), and the blood container lacks a precise positioning device, making it prone to shaking or shifting. The permeable membrane is either completely exposed to air, resulting in unstable temperature, or partially immersed in water, potentially causing contamination or membrane damage. The entire system is typically large and difficult to move, with insufficient temperature control accuracy and stability, and the blood container is easily overturned by the movement of mosquitoes.

[0036] The core problems are inaccurate and unstable temperature control; unstable positioning of blood containers and easy overturning; poor temperature environment of the permeable membrane; and poor portability of the system. Compared with the above-mentioned closest existing technologies, this embodiment has the following significant differences: The integrated temperature-controlled container utilizes a specially designed heating and insulation container with a circular mounting hole 11 with internal threads on its bottom. A micro-thermistor 12 is integrated into the inner wall and connected to a PID temperature controller 13. This achieves high-precision, fast-response closed-loop control of the container's internal temperature, significantly different from the open-loop or low-precision control of a simple water bath. A modular and robust connection structure is employed, incorporating a connecting sleeve 4 as a key interface. This connecting sleeve features external threads 41 that mate with the internally threaded mounting hole 11 on the bottom of the insulation container, enabling a removable screw-in fastening. The connecting sleeve has a specific inner diameter, creating an interference fit with the outer diameter of the lower portion of the blood container 2. The blood container 2 is inserted downwardly into the connecting sleeve 4 and secured thereto, utilizing this interference fit to achieve a tight connection and precise positioning. The bottom, covered with a permeable membrane, extends downward to form an exposed area 5. The position of this exposed area relative to the lower end face of the connecting sleeve (recessed, flush, or protruding) is controllable.

[0037] Through the combination of the above structures, a controlled exposure area 5 is formed at the bottom of the thermal insulation container, which is the actual feeding location of mosquitoes. This embodiment solves the problems of low temperature control accuracy and poor stability through the PID temperature control system. The dual stable structure of threaded connection and interference fit solves the problem of blood containers being prone to shaking, shifting, and even overturning. By tightly integrating the blood container into the bottom of the temperature-controlled container, the exposure area is placed in a relatively stable microenvironment, solving the problem of poor temperature environment, instability, or susceptibility to environmental influences in the permeable membrane area. The container, sleeve, and blood container are detachable for easy carrying, cleaning, and replacement; the overall structure is more compact and stable, improving the modularity, reliability, and portability of the system.

[0038] This embodiment has the following technical effects: extremely high feeding temperature stability, PID temperature control ensures that the blood temperature is accurately maintained at the physiological requirement temperature (such as 38°C), significantly improving the mosquito feeding rate and experimental repeatability. Super strong stability, the threaded connection provides macro fixation, and the interference fit provides micro fixation and sealing, so that the blood container remains motionless when the mosquito swarm feeds, eliminating the risk of overturning. Optimized feeding microenvironment, the exposure area is located near the bottom of the insulation container, and is affected by the thermal environment of the insulation container, with minimal temperature fluctuations, providing a comfortable and stable feeding point for mosquitoes. Convenient operation and portability, the components are easy to disassemble, clean and replace, and the overall structure is compact, which is convenient for transfer and use between different laboratories or field sites. Improved reliability, and stable connections reduce the possibility of operational errors and accidental damage.

[0039] Furthermore, in another embodiment, the bottom of the heating and insulation container 1 is recessed inward around the position of the circular mounting hole 11 to form a coaxial recessed area 15; the depth (H) of the recessed area 15 is 0.5-2 cm; the side walls and bottom walls of the recessed area 15 are directly surrounded by the insulation liquid 16 inside the heating and insulation container 1, so that the recessed area forms an outwardly open mosquito feeding space with a relatively stable temperature.

[0040] Specifically, in a conventional constant-temperature water bath coupled with an open blood container feeding system, the air temperature surrounding the permeable membrane during open feeding is significantly affected by environmental convection, typically fluctuating by >±1°C. Furthermore, conventional flat-surface mounting results in rapid heat loss through metal connectors. Furthermore, mosquito contact stability is poor, requiring mosquitoes to hover above the membrane surface to feed without a guiding structure, thus reducing the success rate of feeding. However, this embodiment further adds the following structural features: The structure of the recessed area 15 consists of a concentric depression machined around the circular mounting hole 11 on the bottom of the heating and insulation container 1. The depth (H) is 0.5-2 cm (preferably 1.5±0.1 cm), and the sidewall inclination angle α is 25-120°. To optimize heat transfer, the sidewalls and bottom of the recessed area 15 directly contact the insulation liquid 16, forming a heat exchange interface. The bottom wall is preferably made of 316L stainless steel (thermal conductivity 16.3 W / m·K) with a thickness of 0.5-2 mm. The ratio of the recessed area depth H to diameter D is set to 0.3-0.3 (optimally 0.33). The recessed area forms an outwardly open, semi-enclosed space with a higher temperature stability than ambient air. This design significantly improves temperature stability. The measured temperature fluctuation within the recessed area (H = 1.5 cm) 3 mm from the membrane surface is ≤±0.2°C, compared to ±1.2°C in a conventional constant temperature water bath.

[0041] This embodiment has the following beneficial effects: thermal efficiency is optimized, the concave structure increases the heat transfer area, and heat loss is reduced compared to a flat structure. The concave sidewalls provide a physical support surface, significantly increasing the contact area for mosquitoes' feet, thereby increasing the success rate of feeding and effectively improving feeding behavior.

[0042] Furthermore, in another embodiment, the lower end surface of the connecting sleeve 4 extends in the inner diameter direction to form an annular stop 42, which is used to abut against the shoulder 21 of the lower outer wall of the blood container when the blood container 2 is inserted downward, thereby limiting the blood container 2 from protruding downward excessively and ensuring the preset exposure height of the permeable membrane 3; Inside the tube wall of the connecting sleeve 4, a thin heat conducting pipe 43 is arranged around the circumference thereof. The thin heat conducting pipe 43 forms a spiral converging flow channel 44 in the area of ​​the connecting sleeve tube wall and the annular baffle 42; The upper end of the thin heat-conducting tube 43 is connected to the outlet of the micro-circulation pump 46 through a flexible insulation pipe 45, and the inlet of the micro-circulation pump 46 extends to the insulation liquid 16 inside the heating and insulation container 1 through another flexible insulation pipe 45. The micro-circulation pump 46 is magnetically coupled driven, and the pump body is located outside the heating and insulation container 1.

[0043] Specifically, this embodiment enhances the functionality of the aforementioned recessed area structure. In the prior art, the extended heat conduction path at the edge of the permeable membrane results in a large temperature gradient, typically reaching 0.5-1.2°C, which reduces mosquito feeding rates. Conventional solutions attempt to compensate for this by increasing temperature overall, but this results in overheating in the central region, even exceeding 40°C, which can cause blood protein denaturation. This embodiment integrates three functional components into the connecting sleeve 4: a mechanical stop structure, with the lower end of the connecting sleeve extending in the inner diameter to form an annular retaining edge 42, whose inner diameter is 0.3-0.5 mm larger than the outer diameter of the lower portion of the blood container 2. The annular retaining edge 42 has a height of 1.0 ± 0.1 mm, designed to precisely abut against the shoulder 21 of the blood container's outer wall, maintaining the exposed height of the permeable membrane within a ±0.2 mm tolerance of the designed position (e.g., recessed / flat / protruding). A distributed microfluidic system is employed. Thin heat conducting tubes 43 are copper alloy capillaries with an outer diameter of 0.8 mm and an inner diameter of 0.6 mm. Six to eight tubes are evenly spaced around the circumference of the connecting sleeve or arranged in a spiral pattern. At the annular baffle 42, the capillaries converge at a 15° helix angle to form a spiral converging channel 44. A closed-loop drive system is employed. A microcirculation pump 46 can be a conventional circulation pump or one driven by magnetic coupling (in the illustration, the microcirculation pump is mounted and fixed to the outer wall of the heated and insulated container). Flexible insulated tubing 45 connects the capillaries. The flexible insulated tubing can be constructed using a three-layer structure: an inner layer of fluororubber for blood corrosion resistance, a middle layer of nano-aerogel insulation with a thermal conductivity of 0.023 W / m·K, and an outer layer of stainless steel braided mesh. The microcirculation pump's inlet line extends 10 mm below the surface of the insulated liquid 16 to prevent air bubbles from being drawn in.

[0044] This implementation addresses the following core issues: uneven feeding caused by temperature differences between the membrane's edge and center; increased aspiration failure due to changes in membrane surface curvature caused by inaccurate mechanical positioning; and the risk of circulatory system leakage. This implementation was tested at an ambient temperature of 25±2°C: Using an infrared thermal imager, the temperature difference across a 20mm diameter membrane surface area was ≤0.05°C, demonstrating excellent surface temperature uniformity. The repeatable positioning error for blood container installation was <±0.15mm, ensuring a constant membrane tension of 12±0.5N / m. The membrane edge temperature was heated from 30°C to 38°C in just 8.3 seconds, demonstrating a very fast thermal response compared to approximately 42 seconds in a conventional constant temperature water bath.

[0045] Furthermore, the axial load-bearing capacity of the annular baffle 42 is designed to be 50N or above, which can withstand the impact load when mosquitoes gather to feed. The taper design of the spiral converging flow channel 44 follows the fluid dynamics equation, pressure drop ΔP = (8μLQ) / (πr 4 ) + ρV 2 / 2, where μ is the dynamic viscosity (0.69 mPa·s for water at 38°C), L is the flow channel length of 12 mm, and Q is the flow rate of 8 mL / min. The calculated pressure drop ΔP is approximately 1.18 kPa, ensuring a flow rate distribution uniformity of >98%.

[0046] Furthermore, in another embodiment, the bottom of the blood container 2 is provided with an inverted conical structure 22, which gradually increases in diameter downward. The inner surface of the inverted conical structure 22 is covered with a biomimetic hydrophilic coating with a contact angle of ≤15°. The coating extends at least 1-2 mm along the inner wall of the blood container to the upper edge of the permeable membrane 3. Specifically, based on the circular heat conduction structure, the bottom geometry of the blood container 2 becomes a key factor affecting feeding efficiency.

[0047] Traditional straight-cylinder containers have approximately 15-22% blood residue, and the edge area of ​​the permeable membrane forms a meniscus effect due to blood tension, resulting in a reduction in the effective feeding area. When the membrane tension increases, it is easy to cause damage. The present embodiment adds the following structural features: an inverted conical structure 22, the cone angle can be designed to be 110°±2° (or other required angles, such as 100°, 120°, 130°, etc.), and the bottom opening of the blood container extends upward by 15mm; the cone wall thickness is 0.8±0.05mm, and the surface roughness Ra≤0.1μm; the ratio of the cone bottom opening diameter to the permeable membrane 3 diameter is 1:1.25, ensuring that the membrane edge extends 2.5mm beyond the cone mouth. Bionic hydrophilic coatings can use existing coating materials such as: BIO-SLICK H101 from Biosaifei Biotechnology, with a contact angle ≤10° and a friction coefficient reduction of >80% under water lubrication conditions; or jAqua from Jemet Coating Technology. TM P-350: A PVP (polyvinyl pyrrolidone)-based hydrophilic, ultra-slip coating specifically designed for medical catheter lubrication. It offers a contact angle of ≤12°, a hemolysis rate of <1%, and is non-cytotoxic according to GB / T 16886.5 testing. Alternatively, a silica-polydopamine nanocomposite (SiO2@PDA) coating is available with a thickness of 500±50nm, a refractive index of 1.38-1.42, a contact angle of ≤15°, and plasma pretreatment. The coating extends from the inner surface of the tapered structure upward to 10mm from the straight section of the container, and extends downward to 1.5±0.2mm from the edge of the permeable membrane.

[0048] The core issues addressed by this embodiment include: low utilization rate due to residual blood, with the residual volume of traditional containers usually >0.3mL. The width of the ineffective feeding area at the edge caused by the meniscus effect is 2-3mm. Capillary blockage caused by the contact angle between blood and the container wall >30°. In this embodiment, 2mL of anticoagulated sheep blood was injected at a constant temperature of 38°C to conduct a residual volume test. The combination of the inverted cone structure and the coating reduced the residual volume to 0.07±0.01mL, while the traditional straight-cylinder container was 0.33mL. The width of the ineffective area at the edge of the membrane was reduced from 2.8mm in the traditional straight-cylinder container to 0.5mm. The coating allows the blood to climb along the cone wall at a speed of 3.2mm / s, while the uncoated one is 0.3mm / s. The conical support reduces the maximum strain of the membrane to 1.2%, while the traditional planar support is >2.5%.

[0049] The optimal 110° cone angle balances the gravitational component (G·sin38°) with the capillary force, allowing the liquid surface to automatically maintain contact with the membrane. The coating structure creates a wetting effect, with an actual contact angle of ≈5°, encouraging blood to spread across the cone. The coating extends downward 1.5mm beyond the membrane edge, eliminating sudden changes in the solid-liquid-gas three-phase line.

[0050] Preferably, the pyramidal structure is injection molded from medical-grade polycarbonate at a mold temperature of 120°C for 15 seconds. To prepare the coating, the substrate is immersed in a dopamine-Tris buffer (pH 8.5) and shaken for 24 hours to form a 50nm polydopamine layer. 200nm SiO2 nanoparticles are deposited by electrostatic self-assembly, followed by a heat treatment at 150°C for 1 hour to enhance adhesion. Finally, an ethylene oxide-modified silane coupling agent is applied to the membrane-container interface using a precision dispensing machine.

[0051] Furthermore, in another embodiment, an independent cup-shaped support frame 23 is provided at the bottom of the blood container 2, comprising: The cup-shaped main structure 23a has an inner diameter that forms an interference fit with the outer diameter of the bottom of the blood container 2, with an interference fit of 0.05-0.1 mm and a height that is 80%-90% of the height of the bottom of the blood container; The regular hexagonal grid structure 23b is located at the bottom of the cup-shaped main structure 23a, with a grid wire diameter of 0.20±0.02mm, an opening side length of 0.8-1.0mm, and an opening ratio ≥65%; A biomimetic hydrophilic coating 23c continuously covers the surface of the grid structure 23b and the inner wall of the cup-shaped main structure 23a, with a coating contact angle of ≤15° and a thickness of 5-10 μm; The permeable membrane 3 is wrapped around the bottom of the blood container 2 and then inserted into the cup-shaped support grid 23 so that the grid structure 23b fits and supports the entire bottom surface of the permeable membrane 3 .

[0052] Specifically, based on the aforementioned inverted cone structure, further efforts are needed to address the deformation of thin permeable membranes (0.05-0.1mm thick) under blood pressure. Traditional nylon meshes have varying coefficients of thermal expansion, which can easily lead to gaps at 38°C.

[0053] The cup-shaped support grid 23 of this embodiment includes: a cup-shaped body 23a, which is made of medical-grade polycarbonate injection-molded standard parts with an inner diameter of Φ25.00±0.05mm, which is suitable for the outer diameter of the bottom of the blood container of Φ24.95±0.03mm; the height of 15mm is 85% of the height of the standard container. The grid structure 23b is made of 316L stainless steel laser-cut mesh with a wire diameter of 0.20mm, a regular hexagonal opening side length of 0.9mm, and an actual opening rate of 68%. The mesh is bonded to the cup body with medical epoxy glue. The bionic hydrophilic coating is the same as the bionic hydrophilic coating mentioned above, with a thickness of about 8±1μm and a contact angle of 12°±2°; it continuously covers the inner wall of the cup body and the surface of the mesh, with a margin of ≤0.2mm. In one assembly process, the permeable membrane is stretched to cover the bottom of the blood container and the edge is fixed with a silicone ring; the cup-shaped support grid 23 is immersed in isopropyl alcohol and ultrasonically cleaned for 5 minutes. After drying in an 80°C oven, it is vertically pressed into the bottom of the blood container (pressure ≤ 5N). The contact pressure between the grid and the membrane is controlled at 0.15-0.25N / cm 2 In this embodiment, mosquito rearing tests (temperature 26±1°C, humidity 70%) revealed a membrane rupture rate of only 0.3 per 1,000 mosquitoes, compared to 2.7 per 1,000 mosquitoes in the control group without a mesh. The residual blood volume was 0.09±0.01mL per 2mL infusion volume. The feeding efficiency was 94% for adult mosquitoes, compared to 78% for the control group without a mesh.

[0054] Example 1 An example of a feeding method of the portable constant temperature piercing-sucking insect feeding system of the present invention is characterized by comprising the following steps: Step 1: Screw the external thread 41 of the connecting sleeve 4 into the circular mounting hole 11 at the bottom of the heating and heat preservation container 1 until the lower end surface of the connecting sleeve 4 is pressed into contact with the bottom surface of the recessed area 15; Step 2: Start the PID temperature controller 13 to heat the insulation liquid 16 in the heating and insulation container to a set temperature of 38±0.3°C at a heating rate of 0.5-1°C / min, and maintain the temperature for at least 10 minutes; Step 3: Turn on the magnetic coupling driven micro-circulation pump 46 to control the flow rate of the insulation liquid 16 through the thin heat conducting tube 43 and the spiral converging flow channel 44 at 5-10 mL / min, and continue to run for at least 5 minutes to stabilize the temperature; Step 4: Cover the permeable membrane 3 with the bottom of the inverted conical opening of the blood container 2 and seal the edges. Insert the blood container 2 vertically downward into the connecting sleeve 4 until the shoulder 21 of the blood container 2 abuts the annular retaining edge 42, securing the blood container 2 in the connecting sleeve 4 through an interference fit. Inject blood preheated to 37-39°C into the blood container 2. Step 5: Allow the mosquito swarm to contact the exposed area 5 of the permeable membrane 3 to feed. When the blood level drops to 1 / 3 of the total height of the container, the flow rate is increased to 12-15 mL / min through the micro-circulation pump 46.

[0055] Specifically, the heating and insulation container uses a constant temperature water bath with a built-in Pt100 thermistor; the PID temperature controller uses a standard industrial controller with a response time of ≤0.1 seconds; the magnetic coupling circulation pump is equipped with a 3.17mm inner diameter pipe; the blood container is a 15mL centrifuge tube, and the permeation membrane uses Parafilm ® M.

[0056] Step 1: When assembling the blood container, apply food-grade silicone grease to the external threads of the 316 stainless steel connecting sleeve and screw it into the pre-threaded hole at the bottom of the insulation container. The threaded hole has a pre-installed silicone gasket. Tighten it tightly to ensure that the lower end is completely aligned with the bottom of the recessed area.

[0057] Step 2: Pour deionized water into the insulation container to the calibration liquid level, with the liquid surface 20 mm from the top; set the parameters of the PID controller: P = 5.0, I = 0.03 min, D = 1.5; perform a step-by-step temperature increase, from the initial temperature of 25°C to 37.5°C, with a heating rate of 0.8°C / min; maintain a constant temperature of 37.5°C for 5 minutes; and then increase the temperature from 37.5°C to 38.0°C at a heating rate of 0.3°C / min; use a thermometer to monitor the surface temperature of the recessed area, and proceed to the next step when the fluctuation is ≤0.1°C within 10 minutes.

[0058] Step 3: When assembling the blood container, stretch the permeable membrane sheet to a thickness of 0.08 ± 0.01 mm, covering the opening of the centrifuge tube in the blood container. Secure the membrane edge with a silicone sealing ring, cut off excess membrane material, and vertically insert the centrifuge tube into the connecting sleeve with an insertion force of ≤15N. Inject preheated blood (preheat sheep blood to 38.0 ± 0.3°C in a water bath). Use a viscometer to confirm a viscosity of 2.8-3.0 mPa·s. Fill the container with 80% of its total volume (9.6 mL for a nominal volume of 12 mL).

[0059] Step 4: Activate the circulation system and start the circulation pump with an initial flow rate of 5 mL / min, corresponding to a speed of 120 rpm; use an infrared thermal imager to monitor the membrane surface temperature difference ΔT in real time: if ΔT ≤ 0.05°C, maintain the current flow rate; if ΔT > 0.05°C, adjust the flow rate according to the formula: target flow rate (mL / min) = 7 + 25 × ΔT (°C). For example, when ΔT = 0.08°C, the flow rate = 7 + 25 × 0.08 = 9 mL / min, and the flow rate upper limit is locked at 15 mL / min, corresponding to a pump speed of 360 rpm.

[0060] Step 5: Manage the feeding process by docking the mosquito cage with the exposed surface of the recessed area; monitor the liquid level by visually checking the centrifuge tube scale every 5 minutes; when the liquid level drops to 6.4 mL (1 / 3 of the total height), increase the pump flow rate to 12 mL / min; and inject 1.0 mL of preheated blood from the same batch into the syringe; feeding ends when the mosquito swarm disperses or the cumulative time is ≥30 minutes.

[0061] Step 6, residual treatment: disassemble the blood container and measure the residual volume; if the residual volume is >0.1mL, inject 5mL of 42℃ normal saline into the syringe; vortex on a vortex shaker at 2000rpm for 10 seconds; repeat the flushing until the effluent is bloodless.

[0062] Comparative Example 1 The traditional feeding method steps are as follows: inject pure water into a constant temperature water bath to 2 / 3 of the volume; set the temperature to 38℃, only open-loop control, no PID algorithm; wait for natural temperature rise to the set value, which takes an average of 22±5 minutes; place a 100mm×20mm culture dish on a float; cover with a permeable membrane, because there is no pre-stretching, the thickness is uneven, and preheated sheep blood is directly poured in, 10mL of which is perfused at a time; the mosquito cage is directly buckled on the top of the water bath; the measured blood temperature fluctuation range is 34.1-36.7℃, and it is directly discarded when the liquid level drops to 3mL; when cleaning, manually scrub the culture dish and float, and soak them in 70% ethanol for 30 minutes.

[0063] Effect experiment The mosquito species was Culex pipiens pallens, 500 per group, with three replicates. The ambient temperature was 26 ± 1°C, and the humidity was 70%. Monitoring equipment used a FLIR T1030sc thermal imager and an EthoVision XT behavioral analysis system.

[0064] Table 1 Test results of Example 1 and Comparative Example Compared with the traditional method of comparative example 1, Example 1 of the present invention has a heating rate of >2°C / min in the traditional method, which easily triggers a heat shock reaction in mosquitoes and has an effective feeding rate of only about 65%. The 0.8°C / min step-by-step heating method of the present invention effectively improves the feeding rate. The edge of the membrane of the traditional device is prone to low temperature, resulting in a decrease in the success rate of blood sucking. The thin heat pipe and dynamic flow control of the present invention eliminate the cold zone, and the effective feeding success rate is >92%. The residual amount of the traditional method is about 0.3mL, accounting for nearly 30% of the total amount. The inverted cone structure and quantitative fluid replenishment of the present invention reduce the residual amount to 0.08mL, accounting for only about 8% of the total amount.

[0065] Example 2 Based on Example 1, step 2 specifically includes: Step 21, controlling the heating rate to 0.8°C / min from room temperature 25°C to 37.5°C by using the PID temperature controller 13; Step 22: The controller starts and maintains a constant temperature of 37.5°C for 5 minutes. The controller automatically switches to PID parameters P=2.0, I=0.05; Step 23, heating the mixture a second time at a rate of 0.3°C / min to 38°C; Step 24: monitor the temperature of the recessed area 15 at a distance of ≤5 mm from the installation position of the permeable membrane in real time through a thermometer sensor. If the fluctuation is ≤0.1°C within 10 minutes, enter the feeding process; if the fluctuation value is greater than 0.1°C within 10 minutes, repeat steps 22-23, and cycle up to 3 times.

[0066] Comparative Example 2 The traditional single-step temperature control method was adopted. A commercially available constant temperature water bath was used to set the target temperature to 38°C. Heating was started directly with a heating rate of >2°C / min. After the temperature reached 38°C, the temperature was maintained constant without any intermediate stabilization steps. Actual monitoring showed that the liquid core thermometer fluctuated between 36.7 and 40.1°C.

[0067] The experimental results of Example 2 and Comparative Example 2 are as follows: Table 2 Anticoagulant sheep blood test As can be seen, the maximum overshoot of the traditional single-step heating method is 1.2°C, while the step heating method of the present invention suppresses the overshoot to within 0.15°C. The traditional method turns off the heating, and the temperature continues to rise by about 0.8°C. However, the 37.5°C insulation stage of the present invention eliminates the thermal inertia of the system, and the temperature continues to rise by only 0.08°C.

[0068] Example 3 Step 3 includes flow rate optimization control: Step 41, initially starting the micro-circulation pump 46 to 5 mL / min; Step 42, obtaining the real-time temperature difference ΔT between the center point and the edge point of the permeable membrane 3 through an infrared sensor; Step 43: If ΔT>0.05°C, the pump speed of the micro-circulation pump is dynamically adjusted according to the formula Vpump=7+25×ΔT (mL / min), and the maximum speed does not exceed 15 mL / min.

[0069] Specifically, the initial pump speed is 5 mL / min, corresponding to a pump speed of 120 rpm; temperature data is collected every 2 seconds, and ΔT = |T 中心 - T 边缘 If ΔT ≤ 0.05°C, maintain the current flow rate. If ΔT > 0.05°C, adjust according to the formula: Target flow rate (mL / min) = 7 + 25 × ΔT (°C). For example, when ΔT = 0.08°C, flow rate = 7 + 25 × 0.08 = 9 mL / min. The final flow rate limit is locked at 15 mL / min, and the pump speed is 360 rpm.

[0070] In steps 3 and 5: Preheat the blood to 38 ± 0.5°C and maintain the temperature for 10 minutes to reduce the viscosity to 2.8-3.0 mPa·s; When the blood level drops to 1 / 3 of the total height, add 0.5-1 mL of preheated blood from the same batch to blood container 2; After a single feeding, the residual blood volume was measured. If it was greater than 0.1 mL, the inner wall of the inverted cone structure 22 was flushed with 42° C. normal saline.

[0071] Specifically, blood was pretreated, a constant temperature water bath with a built-in magnetic stirrer was maintained at 38±0.5°C for 10 minutes, and a viscometer was used to confirm that the viscosity dropped to 2.8-3.0 mPa•s.

[0072] Perform quantitative rehydration. Rehydration is triggered when the liquid level in blood container 2 drops to 1 / 3 of the total height. The rehydration volume is 0.5-1 mL (preferably 0.8 mL). The syringe infusion speed is ≤ 0.2 mL / s. The blood batch should be homologous to the initial blood to avoid antigen differences.

[0073] The residual volume measured by the graduated cylinder was >0.1 mL, and the solution was washed with 42±0.5℃ normal saline. A 10 mL syringe was connected to a blunt needle and spirally sprayed along the inner wall of the inverted cone structure 22 at a flow rate of 5 mL / s.

[0074] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiment. They can be applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily realized.

Claims

1. A portable constant temperature piercing-sucking insect feeding system, characterized in that: include: The heating and insulation container has at least one circular mounting hole with an internal thread on its bottom, and a micro thermistor is provided on the inner wall of the heating and insulation container and is connected to a PID temperature controller for accurately controlling the temperature of the interior space of the container; a blood container having a bottom opening downward; a permeable membrane, sealingly covering the open bottom of the blood container; A connecting sleeve, the outer surface of which is provided with an external thread matching the internal thread of the circular mounting hole; The inner diameter of the connecting sleeve forms an interference fit structure with the outer diameter of the lower part of the blood container; the connecting sleeve is screwed into and fastened to the circular mounting hole at the bottom of the heating and insulation container through an external thread; The blood container is inserted downwardly and fixed in the connecting sleeve, and a tight connection and positioning are achieved through an interference fit structure. At the same time, the bottom of the blood container and the permeable membrane covering it extend downward and are recessed, flat, or protruding from the lower end surface of the connecting sleeve, forming an exposed area at the bottom of the heating and insulation container.

2. The portable constant temperature piercing-sucking insect feeding system according to claim 1, characterized in that: The bottom of the heating and insulation container is recessed inward around the circular mounting hole to form a concave area with a coaxial center. The depth of the recessed area is 0.5-2 cm. The side walls and bottom walls of the recessed area are directly surrounded by the insulation liquid inside the heating and insulation container, so that the recessed area forms an outwardly open and relatively stable temperature mosquito feeding space.

3. The portable constant temperature piercing-sucking insect feeding system according to claim 2, characterized in that: The lower end surface of the connecting sleeve extends in the inner diameter direction to form an annular retaining edge, which is used to abut against the shoulder of the lower outer wall of the blood container when the blood container is inserted downward, thereby limiting the blood container from protruding downward excessively and ensuring the preset exposure height of the permeable membrane; Inside the pipe wall of the connecting sleeve, a thin heat conducting pipe is arranged around the circumference thereof, and the thin heat conducting pipe is arranged on the pipe wall of the connecting sleeve and in the annular baffle area to form a spiral converging flow channel; The upper end of the thin heat-conducting tube is connected to the outlet of the micro-circulation pump through a flexible insulation pipeline, and the inlet of the micro-circulation pump extends to the insulation liquid inside the heating and insulation container through another flexible insulation pipeline. The micro-circulation pump is located outside the heating and insulation container.

4. The portable constant temperature piercing-sucking insect feeding system according to claim 3, characterized in that: The bottom of the blood container is provided with an inverted cone structure, the inverted cone structure gradually increases in diameter downward; The inner surface of the inverted cone structure is covered with a bionic hydrophilic coating, the contact angle of the bionic hydrophilic coating is ≤15°, and the coating extends at least 1-2 mm along the inner wall of the blood container to the upper surface edge of the permeable membrane.

5. The portable constant temperature piercing-sucking insect feeding system according to claim 4, characterized in that: The bottom of the blood container is provided with an independent cup-shaped support grid, including: The cup-shaped main structure has an inner diameter that forms an interference fit with the outer diameter of the bottom of the blood container, and a height that is 80%-90% of the height of the bottom of the blood container; The regular hexagonal grid structure is located at the bottom of the cup-shaped main structure, with a mesh wire diameter of 0.20±0.02mm, an opening side length of 0.8-1.0mm, and an opening ratio ≥65%; A biomimetic hydrophilic coating continuously covers the surface of the grid structure and the inner wall of the cup-shaped main structure, with a coating contact angle of ≤15° and a thickness of 5-10μm; Among them, after the permeable membrane is wrapped around the bottom of the blood container, it is inserted into the cup-shaped support grid as a whole, so that the grid structure fits and supports the entire area of ​​the bottom surface of the permeable membrane.

6. A feeding method of the portable constant temperature piercing-sucking insect feeding system according to any one of claims 1 to 5, characterized in that: The following steps are involved: Step 1: Screw the external thread of the connecting sleeve into the circular mounting hole at the bottom of the heating and insulation container until the lower end surface of the connecting sleeve contacts the bottom surface of the recessed area; Step 2: Start the PID temperature controller to heat the insulation liquid in the heating and insulation container to a set temperature of 38±0.3°C at a heating rate of 0.5-1°C / min, and maintain this temperature for at least 10 minutes; Step 3: Start the micro-circulation pump and control the flow rate of the insulation liquid through the thin heat conduction tube and the spiral converging flow channel at 5-10 mL / min for at least 5 minutes. Step 4: Cover the bottom of the inverted conical opening of the blood container with a permeable membrane and seal the edges; insert the blood container vertically downward into the connecting sleeve until the shoulder of the blood container abuts the annular retaining edge, and secure the blood container in the connecting sleeve through an interference fit; and inject blood preheated to 37-39°C into the blood container; Step 5: Allow the mosquitoes to contact the exposed area of ​​the permeable membrane to feed. When the blood level drops to 1 / 3 of the total height of the container, increase the flow rate to 12-15 mL / min through a micro-circulation pump.

7. The method according to claim 6, characterized in that Step 2 specifically includes: Step 21, controlling the heating rate to 0.8°C / min until reaching 37.5°C by using a PID temperature controller; Step 22, maintaining a constant temperature of 37.5°C for 5 minutes; Step 23, heating to 38°C at a rate of 0.3°C / min; Step 24: monitor the temperature of the recessed area ≤5 mm away from the installation position of the permeable membrane in real time through a sensor. If the fluctuation value is greater than 0.1° C. within 10 minutes, repeat steps 22 to 23.

8. The method according to claim 6, characterized in that Step 3 includes: Step 31, initially start the micro-circulation pump to 5 mL / min; Step 32, obtaining the real-time temperature difference ΔT between the center point and the edge point of the permeable membrane through a sensor; Step 33: If ΔT>0.05°C, the pump speed of the micro-circulation pump is dynamically adjusted according to the formula Vpump=7+25×ΔT, and the maximum value of Vpump does not exceed 15 mL / min.

9. The method according to claim 6, characterized in that In steps 4 and 5: Preheat the blood to 38 ± 0.5°C and maintain the temperature for 10 minutes to reduce the viscosity to 2.8-3.0 mPa·s; When the blood level drops to 1 / 3 of the total height, add 0.5-1 mL of preheated blood from the same batch to the blood container; After a single feeding, the residual blood volume was measured. If it was >0.1 mL, the inner wall of the inverted cone structure was flushed with 42°C saline.

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