The research was funded through the La Trobe University Net Zero Fund, sponsored by Sonepar. The research was conducted under Department of Environment, Land, Water and Planning scientific permit no. 10009741. We thank Martin Steinbauer for comments on an early draft and two anonymous reviewers.

1. Trap construction
NOTE: All of the components required to build the traps can be found in the Table of Materials. Each trap was constructed as shown in Figure 1 and Figure 2 by one person within 2 h.
<ol>
	<li>Use a jigsaw to cut the polycarbonate roofing sheets (8 mm x 610 mm x 2400 mm) into 610 mm x 230 mm sections (Figure 1, item 1 &#38; 2). Then cut a 8 mm center groove halfway up the center of each (610 mm x 230 mm) pane to allow the two panes to slide together to form a cross baffle.</li>
	<li>Slide the crossed baffles snuggly into the plastic funnel opening (Figure 1, item 4) and secure them to the funnel with 20 mm stainless steel angle brackets (Fig 1, item 5b).</li>
	<li>With the angle bracket in place, pre-drill holes and then use M4 screws and nuts with washers (Figure 1, item 6b) to secure the cross baffles to the funnel.</li>
	<li>Again using the jigsaw, cut a piece of the polycarbonate sheeting (230 mm x 305 mm) from remaining sheets and secure with 20 mm stainless steel angle brackets (Figure 1, item 5a) at a 90&#176;&#160;angle to the top of the crossed baffles to form a protective roof (Figure 1, item 3).</li>
	<li>With the angle bracket in place, pre-drill holes and then use M4 screws and nuts with washers (Figure 1, item 6a) to secure the roof to the baffle.</li>
	<li>Trim the funnel spout to a length of ~30 mm with a hacksaw to ensure the sample trays of the automated pet feeder will rotate unobstructed at the programmed intervals.</li>
	<li>Place the automated pet dispenser (Figure 1, item 8) within a 9 L (38 mm diameter) plastic basin (Figure 1, item 7) to protect the samples from weather conditions.</li>
	<li>Drill a 20 mm hole into the top of the 9 L basin and place the funnel spout into the hole to position it directly above the sample tray.</li>
	<li>Using a drill with a hex-head driver bit, secure the plastic basin covering the pet feeder to a 500 mm piece of treated pine fence paling (Figure 1, item 9) with galvanized hex-head screws (Figure 1, item 14).</li>
	<li>To stabilize the entire trap for hoisting into the air via ropes, attach a wooden stake (17 x 17 x 1200 mm, Figure 1, item 10) to the piece of treated pine fence paling (Figure 1, item 9) with an angle bracket and tie wire (Figure 1, items 13, 15 and 16).</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63156/63156fig01.jpg" /> 
Figure 1: Schematic diagram of trap construction. (1 and 2) 610 mm x 230 mm x 8 mm polycarbonate sheets; (3) 230 mm x 305 mm x 8 mm polycarbonate sheet; (4) 24 cm diameter plastic funnel; (5a-b) 20 mm angle brackets; (6a-b) M4 x 15 mm screws, washers &#38; nuts; (7) 38 cm diameter 9 L plastic basin; (8) automated pet food dispenser; (9) 150 mm x 12 mm treated pine paling; (10) 17 mm x 17 mm x 1200 mm wooden stake; (11) 125 mm x 150 mm angle bracket; (12) 16 mm x 16 mm hex-head screws; (13) angle bracket; (14) 16 mm x 16 mm hex-head screws; (15 and 16) wire stabilizer; (17) karabiner, used for lowering and raising into position. <a href="https://www.jove.com/files/ftp_upload/63156/63156fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Trap deployment
NOTE: The traps were attached to trees 6 m above the ground (directly below the experimental or control lights) to capture flying insects (Figure 2). Emptying and collection of traps were done by three people on a single day. Further days can be sampled if required by lowering the trap to remove the collected samples, resetting the pet food dispensers, and placing the trap back in position every three days based on the sampling regime.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63156/63156fig02.jpg" /> 
Figure 2: Low-cost automated flight intercept trap for sampling insects at user-defined time points. (A) Polycarbonate crossed baffles serve as the flight intercept area that allows for collecting insects from all four sides. The polycarbonate roof serves to direct insects downwards and protect the collected samples from the weather. The funnel beneath the flight intercept barrier servers to funnel insects that have collided with the polycarbonate barriers into the collecting trays housed within the circular basin. (B) Trap suspended below experimental light and secured to the tree by a wooden stake and angle bracket. The plywood box below the intercept trap contains a bat detector used to passively record echolocation calls produced by free-ranging insectivorous bats. <a href="https://www.jove.com/files/ftp_upload/63156/63156fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Once at the sampling location, remove the automated pet food dispenser (Figure 1, item 8) from beneath the plastic basin.</li>
	<li>Open up the automated pet food dispenser (Figure 3A) and place foil dishes containing soapy water or a preservative of choice in each food tray (Figure 3B, here 20 mL of propylene glycol was used as a preservative).</li>
	<li>Follow the directions provided with the automated pet food dispenser to set the food tray rotation times. First, set the clock time, then program each pet food dispenser tray. 
	&#8203;NOTE: The automated pet food dispenser rotates food trays (1-6) at pre-programmed times. The 6-meal bowls can be set to open at any time of the day or night, with the trays rotating in sequential order. For this study, the aim was to sample nocturnal and diurnal insects separately. Tray 1 sampled from 8 PM on the first night and then moved to tray 2 at 7 AM the following morning, followed by tray 3 at 8 PM, tray 4 at 7 AM, tray 5 at 8 PM and tray 6 at 7 AM. A delay function allowed for a one-day delay in sampling because it took 2 days to set up all sites, thereby ensuring sampling commenced on the same day/time at all sites.</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63156/63156fig03.jpg" /> 
Figure 3: Automated pet food bowl. (A) Battery-operated 6-meal pet food bowl used to sample insects at user-defined intervals. The food bowls were programmed on alternating schedules to sample nocturnal and diurnal insects. For example, tray 1 opened at 8 PM (nocturnal day 1), tray 2 opened at 7 AM (diurnal day 1), tray 3 opened at 8 PM (nocturnal day 2), tray 4 opened at 7 AM (diurnal day 2), tray 5 opened at 8 PM (nocturnal day 3), and tray 6 opened at 7 AM (diurnal day 3). (B) Lid removed from automated pet food bowl to show the six collection trays. Foil dishes containing propylene glycol as a preservative allowed for the easy removal of the collected insects. <a href="https://www.jove.com/files/ftp_upload/63156/63156fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="4">
	<li>Place the automated pet food dispenser back underneath the plastic basin and secure the basin to the timber fence piece with the galvanized hex-head screws (Figure 1, item 14).</li>
	<li>Attach a rope to the top of the trap with a karabiner (Figure 1, item 17). With the use of a ladder, hoist the trap into position and secure it beneath experimental lights by the karabiner.</li>
	<li>Attach a second wooden stake (17 mm x 17 mm x 1200 mm, Figure 2B) to the tree (or lamp post) with an angle bracket to stabilize the trap in high winds.</li>
	<li>The trap sits on top of the stake; secure it with two large cable ties (Figure 2B).</li>
	<li>To collect insect samples, lower the traps with a rope. Remove the automated pet food dispenser from beneath the plastic basin.</li>
	<li>Remove the lid of the pet food dispenser (Figure 3A) and lift the aluminum trays out to pour the contents into pre-labeled sample vials (Figure 3B).</li>
</ol>

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Despite the automated flight-intercept trap described by Bolliger et al. (2020)8 being well designed and very effective at sampling at user-defined time periods, they are likely to be cost-prohibitive for many researchers. This study shows that passive trapping surveys using automated traps for sub-sampling flying insects at user-defined periods can be carried out on a modest budget. Traps were built to sample at six pre-defined time points by utilizing a commercial pet food dispenser and material...

There are many arthropod sampling techniques1,2,3, but ecologists often have difficulty applying these methods in ways that are appropriate to their research questions (see4). When choosing an appropriate method for sampling insects, ecologists must consider the targeted species, time, effort, and cost involved in different techniques. For example, a common limitation is that it can be logistically challenging to sub-sample during specific time periods over replicated sites to quantify temporal variables which influence species activity, such as changes in weather or circadian activity (but see5). Most passive-survey insect traps are set for long periods (e.g., over multiple days, weeks, or even months), lacking fine-scale temporal resolution1. For surveys targeting specific time periods across multiple replicate sites (such as nocturnal sampling only across distinct sites), a large team may be required to visit sites over multiple days at the same time points (e.g., within 30 min of sunrise and sunset) to collect specimens and reset traps6; otherwise, an automated trapping device is required5,7,8.
There is a growing field of work on the impacts of artificial light at night (ALAN) on insect activity patterns and localized population dynamics9,10; and on the interactions between ALAN and rates of insect predation4,11,12,13. However, to study the impacts of ALAN on nocturnal insect taxa, sampling needs to be confined to nighttime. Several different active light traps have been described and used for automated temporal sampling of nocturnal insects14. Some examples include simple falling disk-type separation devices, where the catch falls into a narrow tube with a disk falling every hour to separate the catch15, or turn-table separation devices that rotate collection bottles at timed intervals7,16,17. These previous automated light traps address the sampling challenges involved with temporal survey requirements but are often large and unwieldy and use outdated or unreliable technology. A new automated passive sampling device was recently developed and tested8. This device utilized a commercially available flight-interception trap paired with a lightweight custom-designed collection device consisting of a turn-table holding sampling cup that allows for collecting trap contents at user-defined intervals8. This new automated trap employs sophisticated programming that can be operated by a smartphone but is prohibitively expensive to build at around EURO 700 (AUD 1,000) per trap8.
Flight intercept traps are one of the most efficient ways to survey flying insects1,18,19 and work on the principle that flying insects fall to the ground when they collide with a vertical surface. Flight intercept traps come in a variety of designs. However, most are typically constructed with a transparent or mesh surface and a collecting container filled with water and/or a preservative. The new trap described here uses a cross vane/baffle type or multidirectional intercept trap20, given that cross baffles have been shown to increase capture rates14,21 and sample insects from all directions. The purpose of this trap is to survey nocturnal flying insects that are attracted to artificial lights. This phototaxis results in insects circling the light source22; hence a multidirectional trap is most suitable.
Described here is a low-cost automated intercept trap that requires no specialist equipment or skills to construct and operate. The trap uses a commercially available automated pet food dispenser and common items available from hardware stores. This design costs less than EURO 66 (AUD 105) per trap to construct (Table 1), making them a viable option for studies requiring temporal sub-sampling across multiple sites simultaneously.

<table><tbody><tr><td>Batteries (C cell) &amp;ndash; 10 pack</td><td>Duracell</td><td>MN1400B10</td><td>https://www.duracell.com.au/product/alkaline-c-batteries/</td></tr><tr><td>Battery operated automated 6 meal pet food bowl &amp;ndash; each</td><td>OEM China</td><td>XR-P006-002</td><td>Automated 6-meal pet food bowls range in price dependent on supplier, for example in the UK they can be purchased for &amp;pound;19 GBP ($36 AUD).</td></tr><tr><td>Galvanised hex-head screws (10-16 x 16 mm) &amp;ndash; 100 pack</td><td>Bunnings Warehouse</td><td>1-311-9151-CTPME</td><td>Bunnings Warehouse is an Australian hardware chain with stores in Australia and New Zealand. Items purchased from Bunnings Warehouse can be found at most hardware stores. https://www.bunnings.com.au/</td></tr><tr><td>Galvanised steel angle bracket (125 x 150 mm) &amp;ndash; each</td><td>Bunnings Warehouse</td><td>AZ11</td><td>https://www.bunnings.com.au/</td></tr><tr><td>Galvanised tie wire (0.70 mm x 75 m) &amp;ndash; per roll</td><td>Bunnings Warehouse</td><td>50218</td><td>https://www.bunnings.com.au/</td></tr><tr><td>Plastic basin (38 cm, 9 L round) &amp;ndash; each</td><td>Ezy Storage</td><td>FBA31541</td><td>https://www.ezystorage.com/product/laundry/basic-accessories/9l-round-basin/</td></tr><tr><td>Plastic funnel (24 cm) &amp;ndash; each</td><td>Sandleford</td><td>Pf24</td><td>https://www.sandleford.com.au/plastic-funnel-24cm</td></tr><tr><td>Stainless steel angle bracket (20 mm) &amp;ndash; 16 pack</td><td>Bunnings Warehouse</td><td>WEB2020</td><td>https://www.bunnings.com.au/</td></tr><tr><td>Stainless steel screws &amp;amp; nuts (M4 x 15 mm) &amp;ndash; 18 pack</td><td>Bunnings Warehouse</td><td>SFA394</td><td>https://www.bunnings.com.au/</td></tr><tr><td>Stainless steel washers (3/16&amp;rdquo; &amp;amp; M5) &amp;ndash; 50 pack</td><td>Bunnings Warehouse</td><td>EBM5005</td><td>https://www.bunnings.com.au/</td></tr><tr><td>Sunlite Polycarbonate roofing sheet (8mm x 610 mm x 2.4 m) &amp;ndash; each</td><td>Suntuf (Palram Industries Ltd)</td><td>SL8CL2.4</td><td>https://www.palram.com/au/product/sunlite-polycarbonate-multi-wall/</td></tr><tr><td>Treated pine paling (150 x 12 mm) &amp;ndash; each</td><td>STS Timber Wholesale P/L</td><td>n/a</td><td>https://www.ststimber.com.au/sts-timber-wholesale-products/fencing</td></tr><tr><td>Wooden stakes (1200 x 17 x 17 mm) &amp;ndash; 10 pack</td><td>Lattice Makers</td><td>n/a</td><td>https://latticemakers.com/product/tomato-stakes-17x17mm-pack-of-10/</td></tr></tbody></table>

low-cost automated flight intercept trap for the temporal sub-sampling of flying insects attracted to artificial light at night

Sampling methods are selected depending on the targeted species or the spatial and temporal requirements of the study. However, most methods for passive sampling of flying insects have a poor temporal resolution because it is time-consuming, costly and/or logistically difficult to perform. Effective sampling of flying insects attracted to artificial light at night (ALAN) requires sampling at user-defined time points (nighttime only) across well-replicated sites resulting in major time and labor-intensive survey effort or expensive automated technologies. Described here is a low-cost automated intercept trap that requires no specialist equipment or skills to construct and operate, making it a viable option for studies that require temporal sub-sampling across multiple sites. The trap can be used to address a wide range of other ecological questions that require a greater temporal and spatial scale than is feasible with previous trap technology.

The traps were trialed in a survey of flying insects attracted to experimental lighting at four bushland reserves across Melbourne, Australia. Sites consisted of either remnant or revegetated bushland surrounded by residential housing and averaged 15 km apart (range 3-24 km) and 45 ha in size (range 30-59 ha). A total of sixteen traps were installed, four at each site, with and without experimental lights (3 lights and 1 control per site), and surveyed for 3 days and 3 nights from 30 March to 2 April 2021. Installing the...

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Low-Cost Automated Flight Intercept Trap for the Temporal Sub-Sampling of Flying Insects Attracted to Artificial Light at Night

To study the impacts of artificial light at night (ALAN) on nocturnal flying insects, sampling needs to be confined to nighttime. The protocol describes a low-cost automated flight intercept trap that allows researchers to sample at user-defined periods with increased replication.

Sampling methods are selected depending on the targeted species or the spatial and temporal requirements of the study. However, most methods for passive ...

low-cost-automated-flight-intercept-trap-for-temporal-sub-sampling

Research

JoVE Journal

Environment

2.5K Views.  La Trobe University. In de studie van lichtvervuiling moet je monsters nemen tussen schemering en zonsopgang. Onze goedkope geautomatiseerde vluchtonderscheppingsvallen maken het mogelijk om vliegende insecten te vangen met door de gebruiker gedefinieerde intervallen. Het kan moeilijk zijn om meerdere locaties tegelijkertijd te bemonsteren, omdat u onderzoekers op elke locatie moet hebben om uw apparatuur tegelijkertijd te starten en te stoppen.Deze val kan worden gebruikt in elke ecologische studie op vli...

Video: Low-Cost Automated Flight Intercept Trap for the Temporal Sub-Sampling of Flying Insects Attracted to Artificial Light at Night

Early Metamorphic Insertion Technology for Insect Flight Behavior Monitoring

Early Metamorphosis Insertion Technology (EMIT) is a novel methodology for integrating microfabricated neuromuscular recording and actuation platforms on insects during their metamorphic development. Here, the implants are fused within the structure and function of the neuromuscular system as a result of metamorphic tissue remaking. The implants emerge with the insect where the development of tissue around the electronics during pupal development results in a bioelectrically and biomechanically enhanced tissue interface. This relatively more reliable and stable interface would be beneficial for many researchers exploring the neural basis of the insect locomotion with alleviated traumatic effects caused during adult stage insertions. In this article, we implant our electrodes into the indirect flight muscles of Manduca sexta. Located in the dorsal-thorax, these main flight powering dorsoventral and dorsolongitudinal muscles actuate the wings and supply the mechanical power for up and down strokes. Relative contraction of these two muscle groups has been under investigation to explore how the yaw maneuver is neurophysiologically coordinated. To characterize the flight dynamics, insects are often tethered with wires and their flight is recorded with digital cameras. We also developed a novel way to tether Manduca sexta on a magnetically levitating frame where the insect is connected to a commercially available wireless neural amplifier. This set up can be used to limit the degree of freedom to yawing &#8220;only&#8221; while transmitting the related electromyography signals from dorsoventral and dorsolongitudinal muscle groups.

We present a novel surgical procedure to implant electrodes in Manduca sexta during its early metamorphic stages. This technique allows mechanically stable and electrically reliable coupling with the neuromuscular tissue to study flight neurophysiology dynamics. We also present a novel magnetic levitation platform for tethered studies of insect yaw.

Early Metamorphosis Insertion Technology (EMIT) is a novel methodology for integrating microfabricated neuromuscular recording and actuation platforms on ...

Behavior

A Precise and Autonomous System for the Detection of Insect Emergence Patterns

Existing systems to measure insect emergence patterns have limitations; they are only partially automated and are limited in the maximum number of emerging insects they can detect. In order to obtain precise measurement of insect emergence, it is necessary for systems to be semi-automated and able to measure large numbers of emerging insects. We addressed these issues by designing and building a system that is automated and can measure emergence of up to 1200 insects. We modified the existing &#34;falling-ball&#34; system using Arduino microcontrollers to automate data collection and expand the sample size through multiple data channels. Multiple data channels enable the user to not only increase their sample size, but also allows for multiple treatments to be run simultaneously in a single experiment. Furthermore, we created an R script to automatically visualize the data as a bubble plot, while also calculating the median day and time of emergence. The current system was designed using 3D printing so the user can modify the system to be adjusted for different species of insects. The goal of this protocol is to investigate important questions in chronobiology and stress physiology, using this precise and automated system to measure insect emergence patterns.

Measurement of insect emergence patterns requires precision. Existing systems are only semi-automated and sample size is limited. We addressed these issues by designing a system using microcontrollers to precisely measure the time of emergence of large numbers of emerging insects.

Existing systems to measure insect emergence patterns have limitations; they are only partially automated and are limited in the maximum number of ...

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Biology

Key Elements of Photo Attraction Bioassay for Insect Studies or Monitoring Programs

Optimized visual attractants will increase insect trapping efficiency by using the target insect&#39;s innate behaviors (positive photo-taxis) as a means to lure the insect into a population control or monitoring trap. Light emitting diodes (LEDs) have created customizable lighting options with specific wavelengths (colors), intensities, and bandwidths, all of which can be customized to the target insects. Photo-attraction behavioral bioassays can use LEDs to optimize the attractive color(s) for an insect species down to specific life history stages or behaviors (mating, feeding, or seeking shelter). Researchers must then confirm the bioassay results in the field and understand the limited attractive distance of the visual attractants.
The cloverleaf bioassay arena is a flexible method to assess photo attraction while also assessing a range of natural insect behaviors such as escape and feeding responses. The arena can be used for terrestrial or aerial insect experiments, as well as diurnal, and nocturnal insects. Data collection techniques with the arena are videotaping, counting contact with the lights, or physically collecting the insects as they are attracted towards the lights. The assay accounts for insects that make no-choice and the arenas can be single (noncompetitive) color or multiple (competitive) colors. The cloverleaf design causes insects with strong thigmotaxis to return to the center of the arena where they can view all the options in a competitive LED tests. The cloverleaf arena presented here has been used with mosquitoes, bed bugs, Hessian fly, house flies, biting midges, red flour beetles, and psocids. Bioassays are used to develop accurate and effective insect traps to guide the development and optimization of insect traps used to monitor pest population fluctuations for disease vector risk assessments, the introduction of invasive species, and/or be used for population suppression.

Photo-attraction bioassay arenas are used to determine the optimal light color(s) to maximize insect attraction; however bioassays and methods are specific to target insect behaviors and habitats. Customizable equipment and modifications are explained for nocturnal or diurnal and terrestrial or aerial insects.

Optimized visual attractants will increase insect trapping efficiency by using the target insect&#39;s innate behaviors (positive photo-taxis) as a means ...

A Wind Tunnel for Odor Mediated Insect Behavioural Assays

Olfaction is the most important sensory mechanism by which many insects interact with their environment and a wind tunnel is an excellent tool to study insect chemical ecology. Insects can locate point sources in a three-dimensional environment through the sensory interaction and sophisticated behavior. The quantification of this behavior is a key element in the development of new tools for pest control and decision support. A wind tunnel with a suitable flight section with laminar air flow, visual cues for in-flight feedback and a variety of options for the application of odors can be used to measure complex behaviour which subsequently may allow the identification of attractive or repellent odors, insect flight characteristics, visual-odor interactions and interactions between attractants and odors lingering as background odors in the environment. A wind tunnel holds the advantage of studying the odor mediated behavioural repertoire of an insect in a laboratory setting. Behavioural measures in a controlled setting provide the link between the insect physiology and field application. A wind tunnel must be a flexible tool and should easily support the changes to setup and hardware to fit different research questions. The major disadvantage to the wind tunnel setup described here, is the clean odor background which necessitates special attention when developing a synthetic volatile blend for field application.

Here, we describe the construction and use of a wind tunnel for odor mediated behavioural assays with insects. The wind tunnel design facilitates the release of odor sources by several methods, with and without visual stimuli. Wind tunnel experiments are important methods to identify behaviorally active volatile chemicals.

Olfaction is the most important sensory mechanism by which many insects interact with their environment and a wind tunnel is an excellent tool to study ...

Flying Insect Detection and Classification with Inexpensive Sensors

An inexpensive, noninvasive system that could accurately classify flying insects would have important implications for entomological research, and allow for the development of many useful applications in vector and pest control for both medical and agricultural entomology. Given this, the last sixty years have seen many research efforts devoted to this task. To date, however, none of this research has had a lasting impact. In this work, we show that pseudo-acoustic optical sensors can produce superior data; that additional features, both intrinsic and extrinsic to the insect&#8217;s flight behavior, can be exploited to improve insect classification; that a Bayesian classification approach allows to efficiently learn classification models that are very robust to over-fitting, and a general classification framework allows to easily incorporate arbitrary number of features. We demonstrate the findings with large-scale experiments that dwarf all previous works combined, as measured by the number of insects and the number of species considered.

We proposed a system that uses inexpensive, noninvasive pseudo-acoustic optical sensors to automatically and accurately detect, count, and classify flying insects based on their flying sound.

An inexpensive, noninvasive system that could accurately classify flying insects would have important implications for entomological research, and allow ...

Bioengineering

Fully Automated Leg Tracking in Freely Moving Insects using Feature Learning Leg Segmentation and Tracking (FLLIT)

The Drosophila model has been invaluable for the study of neurological function and for understanding the molecular and cellular mechanisms that underlie neurodegeneration. While fly techniques for the manipulation and study of neuronal subsets have grown increasingly sophisticated, the richness of the resultant behavioral phenotypes has not been captured at a similar detail. To be able to study subtle fly leg movements for comparison amongst mutants requires the ability to automatically measure and quantify high-speed and rapid leg movements. Hence, we developed a machine-learning algorithm for automated leg claw tracking in freely walking flies, Feature Learning-based Limb segmentation and Tracking (FLLIT). Unlike most deep learning methods, FLLIT is fully automated and generates its own training sets without a need for user annotation, using morphological parameters built into the learning algorithm. This article describes an in depth protocol for carrying out gait analysis using FLLIT. It details the procedures for camera setup, arena construction, video recording, leg segmentation and leg claw tracking. It also gives an overview of the data produced by FLLIT, which includes raw tracked body and leg positions in every video frame, 20 gait parameters, 5 plots and a tracked video. To demonstrate the use of FLLIT, we quantify relevant diseased gait parameters in a fly model of Spinocerebellar ataxia 3.

Fully Automated Leg Tracking in Freely Moving Insects using Feature Learning Leg Segmentation and Tracking FLLIT

We describe detailed protocols for using FLLIT, a fully automated machine learning method for leg claw movement tracking in freely moving Drosophila melanogaster and other insects. These protocols can be used to quantitatively measure subtle walking gait movements in wild type flies, mutant flies and fly models of neurodegeneration.

The Drosophila model has been invaluable for the study of neurological function and for understanding the molecular and cellular mechanisms that underlie ...

Building an Enhanced Flight Mill for the Study of Tethered Insect Flight

Makerspaces have a high potential of enabling researchers to develop new techniques and to work with novel species in ecological research. This protocol demonstrates how to take advantage of the technology found in makerspaces in order to build a more versatile flight mill for a relatively low cost. Given that this study extracted its&#160;prototype from flight mills built in the last decade, this protocol focuses more on outlining divergences made from the simple, modern flight mill. Previous studies have already shown how advantageous flight mills are to measuring flight parameters such as speed, distance, or periodicity. Such mills have allowed researchers to associate these parameters with morphological, physiological, or genetic factors. In addition to these advantages, this study discusses the benefits of using the technology in makerspaces, like 3D printers and laser cutters, in order to build a more flexible, sturdy, and collapsible flight mill design. Most notably, the 3D printed components of this design allow the user to test insects of various sizes by making the heights of the mill arm and infrared (IR) sensors adjustable. The 3D prints also enable the user to easily disassemble the machine for quick storage or transportation to the field. Moreover, this study makes greater use of magnets and magnetic paint to tether insects with minimal stress. Lastly, this protocol details a versatile analysis of flight data through computer scripts that efficiently separate and analyze differentiable flight trials within a single recording. Although more labor-intensive, applying the tools available in makerspaces and on online 3D modeling programs facilitates multidisciplinary and process-orientated practices and helps researchers avoid costly, premade products with narrowly adjustable dimensions. By taking advantage of the flexibility and reproducibility of technology in makerspaces, this protocol promotes creative flight mill design and inspires open science.

This protocol uses three-dimensional (3D) printers and laser cutters found in makerspaces in order to create a more flexible flight mill design. By using this technology, researchers can reduce costs, enhance design flexibility, and generate reproducible work when constructing their flight mills for tethered insect flight studies.

Makerspaces have a high potential of enabling researchers to develop new techniques and to work with novel species in ecological research. This protocol ...

Engineering

A Simple Flight Mill for the Study of Tethered Flight in Insects

Flight in insects can be long-range migratory flights, intermediate-range dispersal flights, or short-range host-seeking flights. Previous studies have shown that flight mills are valuable tools for the experimental study of insect flight behavior, allowing researchers to examine how factors such as age, host plants, or population source can influence an insects' propensity to disperse. Flight mills allow researchers to measure components of flight such as speed and distance flown. Lack of detailed information about how to build such a device can make their construction appear to be prohibitively complex. We present a simple and relatively inexpensive flight mill for the study of tethered flight in insects. Experimental insects can be tethered with non-toxic adhesives and revolve around an axis by means of a very low friction magnetic bearing. The mill is designed for the study of flight in controlled conditions as it can be used inside an incubator or environmental chamber. The strongest points are the very simple electronic circuitry, the design that allows sixteen insects to fly simultaneously allowing the collection and analysis of a large number of samples in a short time and the potential to use the device in a very limited workspace. This design is extremely flexible, and we have adjusted the mill to accommodate different species of insects of various sizes.

Flight in insects is influenced by a number of factors and the propensity to disperse is an important variable in understanding insect ecology and biological control strategies. We describe the construction and use of a simple, relatively inexpensive, and flexible flight mill for measuring parameters of tethered flight in insects.

Flight in insects can be long-range migratory flights, intermediate-range dispersal flights, or short-range host-seeking flights. Previous studies have ...

Neuroscience

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

Low-Cost Automated Flight Intercept Trap for the Temporal Sub-Sampling of Flying Insects Attracted to Artificial Light at Night

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