Tube Caps Research Articles

B-231 Evaluation of the KingFisher Flex for DNA Isolation From FFPE Tissue Utilizing Autolys M Tubes and the MagMAX FFPE DNA/RNA Ultra Kit

Abstract Background Our institution is facing increasing demand for oncology testing that requires high quality DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue. The current DNA extraction workflow involves a time and labor-intensive manual process that limits our ability to accommodate increasing downstream clinical testing volumes. We evaluated the KingFisher (KF) Flex utilizing Autolys M Tubes and the MagMAX FFPE DNA/RNA Ultra Kit as a possible high-throughput, automated DNA isolation workflow option that would decrease hands-on time and be conducive to volume growth without the need for additional staff. Methods DNA from 52 FFPE tissue samples (n=24 unique cases previously extracted by the current “manual method”) were extracted using Autolys M tubes, the MagMAX FFPE DNA/RNA Ultra Kit, and the KF Flex (Thermo Fisher Scientific Waltham, MA) over 4 “automated method” runs. Method modifications to the published procedure were introduced over the runs to determine any effects on DNA quality/quantity. Timings were taken during each run to compare hands-on and total run time against that of the manual method. Precision experiments were performed by extracting 4 replicates of sample on a single run (intra-assay precision) and 2 samples from 2 cases over 2 runs (inter-assay precision). Three cases were processed using varying tissue input levels to assess input-yield correlation. DNA yield was measured using HS dsDNA Qubit (Thermo Fisher Scientific, Waltham, MA). A subset of samples was tested by digital droplet PCR (ddPCR) (n=10), chromosomal microsomal microarray (CMA) (n=1), and next-generation sequencing (NGS) (n=3). Variants and QC metrics of the KF DNA were compared to those of the manual method DNA. Results The average total DNA yield was 3% lower from samples extracted following the manufacturer recommended KF procedure compared to matched samples extracted using the manual method (n=28 samples from 23 unique cases). A CV of 2.8% was observed in an intra-assay experiment (n=4 replicates from 1 case) and 3.0% in an average inter-assay experiment (n=2 replicates from 2 cases). There was a direct correlation between the tissue input amount (4 amounts of varied tissue input) and the total DNA yield (n=3 samples). We observed concordant fractional abundance values for ddPCR samples and concordant variant detection for NGS and CMA samples. We did not see a significant difference in the quality metrics tracked for any of the downstream testing assays. Conclusions This evaluation demonstrated that, as compared to our manual method, the automated method resulted in comparable DNA yields and downstream assay results. The incorporation of this automation would significantly increase our FFPE DNA extraction capacity without requiring additional staff while reducing hands-on time per run by about six hours. We would also be able to reduce ergonomic hazards related to tube cap manipulation, eliminate use of hazardous reagents, and leverage the plate format to better integrate extracted DNA into our clinical sample preparation workflows. These operational benefits highlight the value of automating high-throughput workflows.

Developing a simple and rapid method for cell-specific transcriptome analysis through laser microdissection: insights from citrus rind with broader implications

BackgroundWith the rapid development of single-cell sequencing technology, histological studies are no longer limited to conventional homogenized tissues. Laser microdissection enables the accurate isolation of specific tissues or cells, and when combined with next-generation sequencing, it can reveal important biological processes at the cellular level. However, traditional laser microdissection techniques have often been complicated and time-consuming, and the quality of the RNA extracted from the collected samples has been inconsistent, limiting follow-up studies. Therefore, an improved, simple, and efficient laser microdissection method is urgently needed.ResultsWe omitted the sample fixation and cryoprotectant addition steps. Instead, fresh samples were embedded in Optimal Cutting Temperature medium within 1.5 ml centrifuge tube caps, rapidly frozen with liquid nitrogen, and immediately subjected to cryosectioning. A series of section thicknesses of citrus rind were tested for RNA extraction, which showed that 18 μm thickness yielded the highest quality RNA. By shortening the dehydration time to one minute per ethanol gradient and omitting the tissue clearing step, the resulting efficient dehydration and preserved morphology ensured high-quality RNA extraction. We also propose a set of laser microdissection parameters by adjusting the laser power to optimal values, reducing the aperture size, and lowering the pulse frequency. Both the epidermal and subepidermal cells from the citrus rind were collected, and RNA extraction was completed within nine hours. Using this efficient method, the transcriptome sequencing of the isolated tissues generated high-quality data with average Q30 values and mapping rates exceeding 91%. Moreover, the transcriptome analysis revealed significant differences between the cell layers, further confirming the effectiveness of our isolation approach.ConclusionsWe developed a simple and rapid laser microdissection method and demonstrated its effectiveness through a study based on citrus rind, from which we generated high-quality transcriptomic data. This fast and efficient method of cell isolation, combined with transcriptome sequencing not only contributes to precise histological studies at the cellular level in citrus but also provides a promising approach for cell-specific transcriptome analysis in a broader range of other plant tissues.

Development of a novel Cas13a/Cas12a-mediated 'one-pot' dual detection assay for genetically modified crops

IntroductionGenetically modified (GM) crops have been widely cultivated across the world and the development of rapid, ultrasensitive, visual multiplex detection platforms that are suitable for field deployment is critical for GM organism regulation. ObjectiveIn this study, we developed a novel one-pot system, termed MR-DCA (Multiplex RPA and Dual CRISPR assay), for the simultaneous detection of CaMV35S and NOS genetic targets in GM crops. This innovative approach combined Multiplex RPA (recombinase polymerase amplification) with the Dual CRISPR (clustered regularly interspaced short palindromic repeat) assay technique, to provide a streamlined and efficient method for GM crop detection. MethodsThe RPA reaction used for amplification CaMV35S and NOS targets was contained in the tube base, while the dual CRISPR enzymes were placed in the tube cap. Following centrifugation, the dual CRISPR (Cas13a/Cas12a) detection system was initiated. Fluorescence visualization was used to measure CaMV35S through the FAM channel and NOS through the HEX channel. When using lateral flow strips, CaMV35S was detected using rabbit anti-digoxin (blue line), whilst NOS was identified using anti-mouse FITC (red line). Line intensity was quantified using Image J and depicted graphically. ResultsDetection of the targets was completed in 35 min, with a limit of detection as low as 20 copies. In addition, two analysis systems were developed and they performed well in the MR-DCA assay. In an analysis of 24 blind samples from GM crops with a wide genomic range, MR-DCA gave consistent results with the quantitative PCR method, which indicated high accuracy, applicability and semi-quantitative ability. ConclusionThe development of MR-DCA represents a significant advancement in the field of GM detection, offering a rapid, sensitive and portable method for multiple target detection that can be used in resource-limited environments.

Development of a Test Tube Automatic Sorting Storage Device After Completion of Inspection.

To develop an automatic test tube classification and storage device that sorts test tube boxes by detecting the box height and test tube cap color, improving laboratory efficiency and accuracy. An upper computer uses a No. 1 push rod to allocate test tube boxes to specific branch conveyor belts based on initial sorting criteria. Further sorting is done by the No. 2 push rod, which places boxes into adjustable slots within a seven-layer elevating storage system, categorizing them by day through height adjustment to match the conveyor belt. This invention automates test tube sorting and storage, aiding medical staff in quickly and accurately locating test tubes post-inspection.

Assessment of Preanalytical Cerebrospinal Fluid Handling and Storage Factors on Measurement of Aβ1-42, Aβ1-40, and pTau181 Using an Automated Chemiluminescent Platform.

Standardizing cerebrospinal fluid (CSF) laboratory protocols will improve the reliability and availability of clinical biomarker testing required for prescription of novel Alzheimer disease (AD) therapies. This study evaluated several preanalytical handling and storage factors common to β-amyloid1-42 (Aβ1-42), β-amyloid1-40 (Aβ1-40), and phosphorylated tau (pTau181) concentrations including storage at different temperatures, extended cap contact, various mixing methods, and multiple freeze-thaw cycles. Aβ1-42, Aβ1-40, and pTau181 concentrations were measured using LUMIPULSE G1200 automated assays. Samples were collected in polypropylene tubes of various volumes. Sample cap-contact was evaluated by storing samples in upright and inverted positions at either 4°C for 1 week or -80°C for 1 month. To assess mixing methods, samples were freeze-thawed and mixed by inversion, vortex, horizontal roller, or unmixed prior to assay sampling. The impact of successive freeze-thaw cycles was assessed through freezing, thawing, and analyzing CSF samples. Short-term storage at 4°C did not affect Aβ1-42, Aβ1-40, or pTau181 measurements in any tube type. Tube cap contact affected Aβ1-42 in 2.5 mL tubes and pTau181 levels in 10 mL tubes. No difference was observed between mixing methods. After 4 freeze-thaw cycles, Aβ1-42 significantly decreased but Aβ1-40 remained unchanged. Utilizing the Aβ1-42/Aβ1-40 ratio, Aβ1-42 values normalized, maintaining ratio values within ±5% of baseline measurements. Storage of CSF at 4°C for 1 week or -80°C for 1 month did not significantly affect Aβ1-42, Aβ1-40, pTau181, or associated ratio measurements. Tube cap-contact impacted pTau181 and pTau181/Aβ1-42 values in larger tubes. Mixing methods are equivalent. The Aβ1-42/Aβ1-40 ratio compensates for freeze-thaw variability up to 4 cycles.

An Automatic Implementation of Oropharyngeal Swab Sampling for Diagnosing Respiratory Infectious Diseases via Soft Robotic End-Effectors

The most widely adopted method for diagnosing respiratory infectious diseases is to conduct polymerase chain reaction (PCR) assays on patients’ respiratory specimens, which are collected through either nasal or oropharyngeal swabs. The manual swab sampling process poses a high risk to the examiner and may cause false-negative results owing to improper sampling. In this paper, we propose a pneumatically actuated soft end-effector specifically designed to achieve all of the tasks involved in swab sampling. The soft end-effector utilizes circumferential instability to ensure grasping stability, and exhibits several key properties, including high load-to-weight ratio, error tolerance, and variable swab-tip stiffness, leading to successful automatic robotic oropharyngeal swab sampling, from loosening and tightening the transport medium tube cap, holding the swab, and conducting sampling, to snapping off the swab tail and sterilizing itself. Using an industrial collaborative robotic arm, we integrated the soft end-effector, force sensor, camera, lights, and remote-control stick, and developed a robotic oropharyngeal swab sampling system. Using this swab sampling system, we conducted oropharyngeal swab-sampling tests on 20 volunteers. Our Digital PCR assay results (RNase P RNA gene absolute copy numbers for the samples) revealed that our system successfully collected sufficient numbers of cells from the pharyngeal wall for respiratory disease diagnosis. In summary, we have developed a pharyngeal swab-sampling system based on an “enveloping” soft actuator, studied the sampling process, and implemented whole-process robotic oropharyngeal swab-sampling.

Arabidopsis Root Microbiome Microfluidic (ARMM) Device for Imaging Bacterial Colonization and Morphogenesis of Arabidopsis Roots.

Imaging the spatiotemporal dynamics of host-microbiota interactions is of particular interest for augmenting our understanding of these complex systems. This is especially true of plant-microbe interactions happening around, on, and inside plant roots where relatively little is understood about the dynamics of these systems. Over the past decade, a number of microfluidic devices have been developed to grow plants hydroponically in gnotobiotic conditions and image morphogenesis of the root and/or dynamics with fluorescently labeled bacteria from the plant root microbiome. Here we describe the construction and use of our Arabidopsis Root Microbiome Microfluidic (ARMM) device for imaging fluorescent protein expressing bacteria and their colonization of Arabidopsis roots. In contrast to other plant root imaging devices, we designed this device to have a larger chamber for observing Arabidopsis root elongation and plant-microbe interactions with older seedlings (between 1.5 and 4weeks after germination) and a 200μm chamber depth to specifically maintain thin Arabidopsis roots within the focal distance of the confocal microscope. Our device incorporates a new approach to growing Arabidopsis seedlings in screw-top tube caps for simplified germination and transfer to the device. We present representative images from the ARMM device including high resolution cross section images of bacterial colonization at the root surface.

ElectrochemCap: an integrated detection for loop-mediated isothermal amplification reactions.

Loop-mediated isothermal amplification (LAMP) of genetic materials has emerged as a powerful molecular biology technique with great potential to be a standard point-of-care (POC) technique. This method has found several applications, but it still presents challenges for its direct on-site application, particularly in terms of integrated reaction and detection systems and the risk of carryover contamination. In this work, we propose an innovative solution - an electrochemical microcentrifuge tube cap (ElectrochemCap) based on a screen-printed electrode, a 3D printed adapter and an adhesive layer - which integrates the amplification reaction and its subsequent electrochemical detection in a single device. The design, fabrication, and electrochemical characterization of the ElectrochemCap are reported here, demonstrating its suitability for LAMP detection. Rapidly emerging technologies, such as 3D printing or xurography, are the basis of a prototype that has been validated for the detection of SARS-CoV-2 using reverse transcription LAMP (RT-LAMP), achieving results comparable to those obtained by gold-standard RT-qPCR. Moreover, we have explored the versatility of the ElectrochemCap presenting several additional designs (for containers with different volumes, shapes and materials, as well as multiplexed approaches), expanding its potential applications. Overall, the ElectrochemCap represents an affordable, versatile, and marketable innovation for integrated quantitative electrochemical detection, with enormous possibilities in bioelectroanalytical procedures and portable laboratory setups.

Tiny swabs: nasal swabs integrated into tube caps facilitate large-scale self-collected SARS-CoV-2 testing.

The COVID-19 pandemic spurred the development of innovative solutions for specimen collection and molecular detection for large-scale community testing. Among these developments is the RHINOstic nasal swab, a plastic anterior nares swab built into the cap of a standard matrix tube that facilitates automated processing of up to 96 specimens at a time. In a study of unsupervised self-collection utilizing these swabs, we demonstrate comparable analytic performance and shipping stability compared to traditional anterior nares swabs, as well as significant improvements in laboratory processing efficiency. The use of these swabs may allow laboratories to accommodate large numbers of sample collections during periods of high testing demand. Automation-friendly nasal swabs are an important tool for high-throughput processing of samples that may be adopted in response to future respiratory viral pandemics.

Phenology and thallus size in a non-native population of Gracilaria vermiculophylla.

Phenology, or seasonal variation in life cycle events, is poorly described for many macroalgal species. We describe the phenology of a non-native population of Gracilaria vermiculophylla whose thalli are free-living or anchored by decorating polychaetes to tube caps. At a site in South Carolina, USA, we sampled 100 thalli approximately every month from January 2014 to January 2015. We assessed the reproductive state and measured thallus size based on wet weight, thallus length, and thallus surface area from herbarium mounts. Because life cycle stage cannot be assigned using morphology, we implemented a PCR assay to determine the life cycle stage-tetrasporophyte, female gametophyte, or male gametophyte-of each thallus. Tetrasporophytes dominated throughout the year, making up 81%-100% of thalli sampled per month. Reproductive tetrasporophytes varied between 0% and 65% of monthly samples and were most common in warm summer months (July through September) when thalli also tended to be larger. The vast majority of the reproductive thalli were worm-anchored and not fixed to hard substratum via a holdfast. Thus, free-living thalli can be reproductive and potentially seed new non-native populations. Given G. vermiculophylla reproduction seems tied closely to temperature, our work suggests phenology may change with climate-related changes in seawater temperatures. We also highlight the importance of understanding the natural history of macroalgae to better understand the consequence of range expansions on population dynamics.

PLASTIC DEBRIS USAGE BY TUBE-BUILDING POLYCHAETE Diopatra cuprea COMPLEX

In this short note the plastic debris usage by tube-building polychaete Diopatra cuprea complex in the tube caps decoration is reported for the first time. Soft plastics attached to tube caps were recorded at a tidal flat in the Sergipe River estuary, northeastern Brazil. This interaction probably reflects an adaptation of this polychaete to a highly polluted environment. Keywords: pollution, soft plastics, tube caps, decoration.

Development of an Endotracheal Tube Cap for Oxygen Delivery During Intubation

AbstractEndotracheal (ET) Intubation is a medical procedure whereupon a physician or trained personnel inserts a breathing tube into a patient's mouth, through their vocal cords, and into their trachea. Intubation can be lifesaving when a patient cannot breathe on their own. Intubations are performed routinely, with approximately 15 × 106 performed annually just in the operating room (OR) with an additional 650,000 intubations in the wider hospital environment. Intubation is a complex, dynamic, and at times difficult procedure with major consequences if delayed and/or if the procedure fails. Complications for intubations outside of the operating room are reported as high as 27%, with the most common being hypoxia, or low oxygen levels. We have developed a simple, sterile attachment that directs oxygen down endotracheal tubes during intubation. Benchtop studies have demonstrated acceptable pressures well below those leading to barotrauma. In animal studies, the device has been shown to significantly reduce hypoxia; thereby increasing the time a medical provider has to safely perform the procedure. While further development is warranted, as well as additional testing both in vitro and in vivo, the cap assembly appears to provide a viable solution to a persistent and dangerous problem in medicine.

The effect of sucrose concentrations and different types of tube cap on in vitro growth of Dahlia (Dahlia sp.) using vermiculite as substrate

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Colorimetric detection of viable antibiotic resistant Enterococcus mediated by cordless operation of reverse transcription loop-mediated isothermal amplification

In this study, we applied a tube-based reverse transcription loop-mediated isothermal amplification technique using preloaded amplification and detection reagents for simple screening of viable vancomycin-resistant Enterococcus in a cordless manner. We adopted an mRNA-based approach to detect live Enterococcus in vancomycin-treated cultures. We used agarose to preload and store all reagents for amplification and detection inside the tube, which could achieve on-site isothermal nucleic acid amplification and detection in less than 1 h without using sophisticated instruments. Moreover, the use of a portable insulated water tumbler eliminated the need for electricity, which is usually important in nucleic acid amplification-based assays. The water tumbler acted as a heat source to supply a stable heat required for the amplification reaction, which could last up to 45 min. In addition, colorimetric detection was realized using pH-based methods. The detection was triggered by shaking the tube so that the amplified solution was reacted with phenolphthalein embedded in the tube cap. The introduced one-pot strategy has many advantages such as easy and cordless operation, low cost, disposability, and less chance of contamination because the amplification and detection occur in a closed system. The system could have a great impact on nucleic acid analyses in instrument-free and low-resource areas.

Photoactivatable CRISPR/Cas12a Strategy for One-Pot DETECTR Molecular Diagnosis.

As a golden partner of recombinase polymerase amplification (RPA), CRISPR/Cas12a has been proven to solve the false-positive problem caused by nonspecific amplification perfectly; meanwhile, its trans-cleave activity has further enhanced the sensitivity. However, the solution transfer operation after tube cap opening greatly increases the risk of aerosol contamination of amplicon, which is inconsistent with point-of-care (POC) diagnostics requirements. This study proposes a photoactivated CRISPR/Cas12a strategy to achieve one-pot high-sensitivity nucleic acid detection. Using photocleavable complementary ssDNA to block crRNA, RPA amplification can smoothly pass through the exponential interval without being affected by activated Cas12a in the critical early stage. After enough amplicons were produced, the Cas12a test was activated by short bursts of ultraviolet radiation at 365 nm. This one-pot method achieved a sensitivity of 2.5 copies within 40 min. This simple and sensitive one-pot method can effectively avoid amplicon contamination and lower the threshold for molecular diagnostics in POC.

Native tube-building polychaete prefers to anchor non-native alga over other macrophytes.

Novel facultative mutualisms that develop between native and non-native ecosystem engineers can lead to the retention of the non-native partner. In some cases, behavior plays an additional, but less understood, role in the development and persistence of mutualisms. In soft-sediment marine habitats along the western Atlantic, the native decorator worm Diopatra cuprea anchors the non-native red alga Gracilaria vermiculophylla to its tube cap in a mutualism. To understand whether the worm's usage of G. vermiculophylla could represent a preference, we first surveyed the species composition of macrophytes affixed to worm tube caps at three sites in coastal Virginia, USA using transect and quadrat sampling. These unmanipulated field surveys supported previous work revealing variable, but often high frequencies (31-98%) of D. cuprea decoration with G. vermiculophylla. We next used field manipulations and controlled laboratory experiments to test the consistency of individual D. cuprea decoration with G. vermiculophylla versus three common macrophytes (Ulva sp., Agardhiella sp., and Spartina alterniflora) found in our field surveys. Twenty-four hours after removing the worm's tube cap in the field, D. cuprea decoration was dominated by both G. vermiculophylla (39.6%) and S. alterniflora (25.9%). When provided a choice of macrophytes in the laboratory, individual D. cuprea consistently decorated with G. vermiculophylla (58.7%) over the other macrophytes, showing a preference for the non-native macrophyte. Our study suggests that preference can drive strong and steadfast interactions between native and non-native organisms, facilitating the latter's persistence and spread, change available habitat, and alter community interactions.

An algorithm-assisted automated identification and enumeration system for sensitive hydrogen sulfide sensing under dark field microscopy.

Hydrogen sulfide (H2S) is an active physiological molecule, and its intracellular level has great significance to life functions. In this study, an effective and sensitive method was developed for H2S sensing with dark field microscopy (DFM). The proposed method employed AuNPs as the signal source, DFM as the readout system, and an intelligence algorithm as the image processing and output systems, respectively. The AuNP surface was modified with azido and alkynyl in advance, and then added into a tube cap. As the H2S evaporated from the solution and selectively reduced azido to amino, the click chemistry reaction was inhibited, which resulted in the AuNPs being well dispersed in the solution; otherwise, AuNP aggregation occurred. The scattering colour of single AuNPs could be easily distinguished from that of AuNP aggregations with DFM, and the number or ratio of single AuNPs could also be easily obtained by the custom algorithm. The results showed that the H2S content could be linearly analyzed in a range from 2-80 μM. Furthermore, the proposed sensing strategy has been applied for H2S detection in cell lysate. Compared with the traditional colorimetric method, the results showed no significant difference, indicating the good prospects of the algorithm and proposed H2S sensing method.

Fast seamless joining of SiCw/Ti3SiC2 composite using electric field‐assisted sintering technique

AbstractA pair of Ti3SiC2 reinforced with SiC whiskers (SiCw/Ti3SiC2) composites was successfully joined without any joining materials using electric field‐assisted sintering technology at a temperature as low as 1090°C (Ti) and a short time of 30 s. The microstructure and mechanical properties of the obtained SiCw/Ti3SiC2 joints were investigated. The solid‐state diffusion was the main joining mechanism, which was facilitated by a relatively high current density (~586 A/cm2) at the joining interface. The shear strength of the sample joined at 1090°C was 51.8 ± 2.9 MPa. The sample joined at 1090°C failed in the matrix rather than at the interface, which confirmed that a sound inter‐diffusion bonding was obtained. A rapid and high efficient self‐joining process may find application in the case of SiCw/Ti3SiC2 sealing cladding tube and end cap.

Automated Device for Uncapping Multiple-Size Bioanalytical Sample Tubes Designed to Reduce Technician Strain and Increase Productivity.

Technicians in a commercial laboratory manually uncap up to 700 sample tubes daily in preparation for bioanalytical testing. Manually twisting off sample tube caps not only is a time-consuming task, but also poses increased risk for muscle fatigue and repetitive-motion injuries. An automated device capable of uncapping sample tubes at a rate faster than the current workflow would be valuable for minimizing strain on technicians' hands and saving time. Although several commercial sample tube-uncapping products exist, they are not always usable for a workload that uses a mix of tube sizes and specific workflow. A functioning uncapping device was developed that can semi-automatically uncap sample tubes with three different heights and diameters and was compatible with the workflow in a commercial laboratory setting. Under limited testing, the average success rate with uncapping each of the three sample tube sizes or a mix of them was 90% or more, more than three times faster than manual uncapping, and met standard acceptance criteria using mass spectrometry. Our device with its current performance is still a prototype, requiring further development. It showed promise for ergonomic benefit to the laboratory technicians, however, reducing the necessity to manually unscrew caps.

Fighting Behaviour of Male Onion Thrips, Thrips tabaci (Thysanoptera: Thripidae) Lineages

Onion thrips, Thrips tabaci Lindeman, 1889 (Thysanoptera: Thripidae) has three distinctive reproductive modes: arrhenotokous, thelytokous and deuterotokous. This experiment was focused on the arrhenotokous leek-(L1) and tobacco-associated (T) T. tabaci lineages. These two lineages are distinctively varied genetically and in host adaptations. L1 and T lineages perform better on leek and tobacco plants, respectively. Fighting occurs between males when they compete for food, mating, and oviposition sites. The aim of this research was to examine the fighting behaviour and characterize the fighting elements of males in L1 and T lineages. The experiment was performed in the laboratory by using a Euromex VC.3036 video camera and each experiment was recorded for a duration of 10 minutes. Transparent PCR tube caps formed the arena to observe the fighting interactions in both lineages. A total of 40 video recordings have been observed and each recording had a different arena. The fighting performance was observed at 2, 5, 8, 10, 12 days old specimens in four replications. This result has shown L1 lineage had better fighting performance and a more aggressive fighting ability than T lineage. Antennal bouts, jumping, flipping, stabbing and pitching are the most commonly observed fighting elements.