Instructions

This is the sampling protocol that has been refined together with our sampling teams all over the world. Please read the instructions carefully well before starting sampling. 

There will be more detailed user manuals that come with the camera traps and the audio recorders. The data collection will be supported by a simple smartphone app, which is being developed and will also have a separate user manual. 

Preliminary plan, to be finalized together with participants:

  • Six years from September 2020 to end of 2025
  • Setup: Selecting natural and urban locations and setting up a 100metre x 100metre plot in one of these. Installing Malaise trap, cyclone sampler, about 5 cameras and about 5 audio samplers (attached to trees).
  • Yearly: moving all sampling equipment from natural to urban location or vice versa
  • Eight times a year during growing season: taking eight soil samples
  • Weekly: one longer visit: changing batteries on all devices (Cyclone sampler runs on car battery), changing sampling tubes on cyclone sampler and Malaise trap, changing memory cards on audio samplers and cameras. Logging and labelling samples. Two quick visits on the two following days to change the tube on the cyclone sampler.
  • At least twice a year: shipping samples to us
Global sampling plan of project LIFEPLAN, University of Helsinki.

Figure 1: Global sampling design. Locations of dots represent the even coverage we are aiming for, not specific desired locations.

 

Hierarchical sampling design for project Lifeplan, University of Helsinki.

Figure 2: Hierarchical sampling design

 

The LIFEPLAN sampling scheme contains 100 global locations (Fig. 1), 50 national locations (25 in Sweden and 25 in Madagascar, (Fig. 2), and two sets (in Sweden and in Madagascar) of hierarchical designs that cover the scales from 100 meters to 100 km (Fig. 2 insets). Here we describe only the global and the national sampling schemes (Fig. 1 and Fig. 2 maps), since the hierarchically lower levels (Fig. 2 shaded boxes) will be sampled by LIFEPLAN staff.

As described in section What happens after you join?, the only difference between the global and national sampling schemes is that at the global scale, we will also explore the impacts of urbanization. Hence, for each of the 100 locations spread across the world, we switch between an urban and a natural location on a yearly basis, whereas at the national level, locations remain fixed.

How “urban” the urban site should be, and how “natural” the natural site should remain depends on the types of environments available in the particular area. In some places, the urban location can mean the neighborhood of a village of 100 inhabitants. What matters is that the two sites within the urban-natural site pair differ substantially in their level of naturalness, that they are located some 20-50 km from each other, and that in terms of local conditions, they resemble each other as much as possible beyond their respective level of naturalness.

At each sampling location, you should establish a one-hectare (100 m x 100 m square) sampling plot. The plot should be placed in a habitat type that represents vegetation typical to the area, e.g. a common forest type.

Five types of sampling will be conducted within this area: audio recording, camera trapping, cyclone sampling, Malaise trapping, and soil sampling (Fig. 2). The sampling plot will have a single cyclone sampler and a single Malaise trap, placed near the center of the plot. The sampling plot will also have five camera traps and five audio recorders. These you should place as five pairs, one near the center of the plot and one at each corner. Each pair of a camera trap and an audio recorder should be attached to the same tree. 

Equipment provided

  • Five AudioMoth 1.0 recorders with waterproof cases
  • AA Batteries
  • Battery charger
  • microSD cards
  • Memory card reader
  • External hard drive

Setup

The audio recorders will be pre-programmed to record at set periods, to be determined later. Attach the five audio recorders to the same trees as the five camera traps, but higher up. Four recorders should be at the four corners of your one-hectare plot, and one in the middle of the plot. An accuracy of about 10 metres is fine. If there are no trees, please put up sturdy posts where they can be mounted at about 1 or 2 metre height. Please put up appropriate signage by the recorders according to local legislation. Appropriate signage will likely depend on whether you are on private property or in a public space. Note that legislation for audio recording is more strict than for cameras in many countries.

Weekly sampling

At each audio recorder, use the LIFEPLAN app to scan the QR code on the recorder, and then the QR code on the SD card you are about to insert. Remove the previous SD card and replace with a new one. Change the batteries. Set local date and time. Put the recorder back in its tree. 

When you return from the field, insert the full memory card into the card reader, plugged into your computer. Upload the files through our web interface . We will provide detailed instructions for uploading at a later time. Also copy the files from the memory card onto the external hard-drive for a local backup. When you have safely uploaded and backed up the files, erase the SD card so you can use it again next week.

Equipment provided

  • Camera traps: new custom model "4.0C" from Wildlife Monitoring Solutions 
  • Cable ties
  • Batteries
  • Battery charger
  • SD cards
  • Memory card reader
  • External hard drive

The camera traps

The camera traps are motion triggered day and night using a passive infrared sensor and will be set to operate continuously at each sampling plot.

First deployment

Four camera traps will be set up in each corner of your one-hectare plot, and one in the middle of the plot. An accuracy of about 10 meters is fine. The camera traps will be mounted on trees. If there are no suitable trees, please put up sturdy posts (e.g. tree posts used for fences or similar). The tree/post should be sturdy enough to withstand weather and not sway in windy conditions (this might trigger the camera).

Mount the cameras at a height of 0.5 metres from the ground. If there is snow, move the camera up or down accordingly so it starts each week 0.5 m above the snow. If needed, cut down vegetation in front of the camera to give visibility and to reduce false triggering by vegetation moving in the wind. If you feel this camera height is impossible at your location, contact lifeplan@helsinki.fi to work out a solution.

Use the mounting strap to mount the camera to the tree. If your site is insecure and prone to theft, let us know at lifeplan@helsinki.fi and we can supply Python cables for added security. The camera traps should be faced north in the northern hemisphere and south in the southern hemisphere to avoid glare from the sun. Try to choose a place with the least amount of obscuring vegetation, and don't point the center camera at the cyclone sampler and Malaise trap. 

When you have found a good spot, set up the camera with the LIFEPLAN pre-settings. The camera trap should be perpendicular to the ground in front of the camera. This will mean a bit of extra work if the tree is on a slope, as the camera will need to be angled either upwards or downwards (down is better, to avoid glare). This can be done with sticks or stones that are found at the site. When you have deployed the camera trap, note down for yourself where the camera trap is positioned so you easily find it again. Make a note if there are any interesting features in the immediate area that might affect detection (for example game trails). Once the camera is running with fresh batteries and memory cards, secure the latch with a cable tie to prevent tampering.

Please put up appropriate signage by the camera traps according to local legislation. The signage may depend on whether you are on private property or a public place.

Good things to keep in mind:

  • Try to choose a tree that looks like it will keep standing the entire study period. The cameras should, if possible, be mounted in the same spot throughout the project.
  • If you are in an open habitat and you put up a post, keep in mind that it might be seen as an excellent scratching post for local wildlife. So try to make sure the post is as sturdy as possible! 
  • Keep an eye out for nearby nests (like ants or termites) that could make maintenance difficult for you. 
  • Keep in mind that the audio recorders will be mounted on the same trees/posts, so there should be some free space above the cameras.

Weekly sampling

Make sure you have enough charged batteries for all the cameras, and that you have a system in place to separate empty batteries from the charged ones. When arriving at a camera, do a quick scan of the camera and the area to check if anything is out of the ordinary. You will be able to note down any anomalies in the LIFEPLAN app (for example if the camera is facing in the wrong direction or is lying on the ground). 

Cut off the cable tie and open the camera. Remove the previous SD card and store it in a safe way until you return from the field. Use the LIFEPLAN app to scan the QR code on the camera trap and then the QR code on the new SD card. Change the batteries and insert the new SD card. Put the camera trap back in its tree, making sure it is facing the same direction as before and is placed at the same height. Secure the latch with a cable tie to prevent tampering.

When you return from the field, insert the full memory card into the card reader, plugged into your computer. Upload the files through our web interface. There will be more specific instructions for uploading later on. Also copy the files from the memory card onto the external hard-drive for a local backup. When you have safely uploaded and backed up the files, erase the SD card so you can use it again next week.

Equipment provided

Equipment you provide

  • 2 x 12 V car batteries, or mains power
  • Freezer (preferred) or refrigerator for sample storage

Setup

Select a place that

  • Is roughly in the middle of your one-hectare plot.
  • Is easy to get to: remember car batteries are heavy. At the same time,  the sampler should be at least 15 metres from any roads to avoid dust from traffic.
  • Has room for the sampler’s wind plate to rotate freely.
  • Has room for the battery to sit next to it protected from rain OR has access to a power supply. If possible, a roof over the cyclone sampler, at 2 metre height, with an area of a couple of square metres, helps protect from rain and will not disrupt sampling.
  • Has level, firm ground: the legs of the sampler should not sink in the ground.
  • Place the sampler so that it is level. Please use a spirit level. This will enable the wind plate to rotate and direct the sampler into the wind.

The sampler comes with crocodile grips for connecting to a 12 V car battery, or you can use the adapter to connect to your mains power supply.

Weekly sampling

(The weekdays do not have to be exactly Monday-Wednesday, as long as they are the same three consecutive days every week.)

On Monday morning, bring a fully charged 12 V car battery. Use the LIFEPLAN app to scan the QR code on the cyclone sampler and then the QR code on the new tube you will put in. In the app, select “replace” to start a new sample. Remove and open the cyclone unit. Wearing a glove, screw in the empty tube you just scanned. Store the cap safely. Reassemble and replace the cyclone unit. Connect the battery and listen for the vacuum pump running.

24 hours later, bring another fully charged 12 V car battery. Disconnect the old battery. Remove and open the cyclone unit. Wearing a glove, remove and close the first tube. Take care to use its original lid. Use the LIFEPLAN app to scan the QR code on the cyclone sampler (select “collect” in the app)  and then the QR code on the new tube you will put in (select “replace” in the app). Store the new cap safely. Reassemble and replace the cyclone unit. Connect the new battery and listen for the vacuum pump running. Take the old battery with you for recharging.

Another 24 hours later, disconnect the battery. Remove and open the cyclone unit. Wearing a glove, remove and close the second tube. Take care to use its original lid. Use the LIFEPLAN app to scan the QR code on the cyclone sampler (select “collect” in the app). Reassemble and replace the cyclone unit. Take the old battery with you for recharging.

In addition, the car battery providing the energy for the sampler needs to be charged between Wednesday and Monday. The collected vials are stored in a freezer or refrigerator, and posted to the LIFEPLAN project team in Uppsala, Sweden at least twice per year.

Alternative arrangements

If you have a long travel time to the site and need to reduce the number of weekly visits, you can

  1. Set up a timer (to be provided upon special request), that cuts off power to the sampler after 24 hours: this way you don’t have to do the third visit but just collect the tube the following Monday.
  2. Only do one sample a week: make sure you contact us before deciding to do this so we can make sure there is no other way. The second sample is valuable, so we want to collect it if at all possible.

If you need a timer or to discuss taking only one sample a week, please email lifeplan@helsinki.fi

Sampling protocol

It is critical that we employ standardized operating procedures for the Malaise trapping. Our coordinated efforts will ensure specimen preservation for sequence analysis and high data quality, permitting the comparison of sites at a global scale. For global standardization with the BIOSCAN initiative, of which LIFEPLAN is a part, we have adopted all the same basic protocols as used in the Global Malaise Program. As a key difference, while in the GMP processing was done on individual arthropods, LIFEPLAN will be based on bulk processing (metabarcoding) of samples. This solution is necessary because of the enormous size of the data to be generated vs the finite resources. At the same time, there is funding to barcode at least 1,000 individual arthropods per site. Through this dedicated initiative, we will help build national reference libraries, as well as contribute to worldwide taxonomic initiatives and to barcode-based identification resources.

Equipment provided:

  • Malaise trap kit including assembly instruction sheet
  • 52x 500ml Collection bottles with QR codes; external and internal labels
  • Pipette rubber bulbs
  • Decanting lid
  • Nitex filters

Equipment you provide:

  • 95% ethanol for preserving samples
  • Ethanol waste container
  • Cooler box: styrofoam or similar
  • Freezer (preferred) or refrigerator for sample storage
  • Clean duster

Setup

As our standard Malaise Traps, we use the model “ez-Malaise Trap” manufactured by MegaView Science Co. (Bugdorm), with trap design and kit contents shown at the Bugdorm and BioQuip web pages. Compared to the commercially available design, we have made a few modifications: we use Nalgene bottles and barbed tent pegs, and we have removed the "moth excluder device" (a triangular piece of cloth with small holes, used to keep out bigger insects when focusing on Diptera and Hymenoptera). The support poles at the front and back of the traps have also been upgraded to a sturdier material than in the trap kits sold online. To reduce wear by the pole ends on the cloth pockets holding them in place, we provide pipette rubber bulbs (to be mounted by the LIFEPLAN teams on the ends of the poles before sticking them into the pockets).

The trap should be placed near the center of the one-hectare sampling plot. When mounting, make sure that the collecting part of the trap (i.e. the trap head) is directed towards the equator (north in the Southern hemisphere and south in the Northern hemisphere), and that this is the highest part of the trap (i.e. avoid placing the trap on a downhill slope with the collecting part lowest) (Figure 3). This is because the trap design depends on insects moving towards the highest and lightest part of the trap. When possible, position the trap at a right angle to an insect flight line, in areas with low undergrowth; forest edges or clearings and elevated sites are recommended.  

 

Positioning of Malaise traps relative to insect flight paths. Project Lifeplan, University of Helsinki.

Figure 3. Positioning of Malaise trap relative to landscape edge and southerly direction

Consider possibilities of wildlife disturbance and/or human vandalism – try to avoid either scenario as much as possible; the trap may be relocated if consistent issues persist after deployment. Ensure that all proper specimen collecting permissions are obtained (i.e. from local authorities, property owners, etc.). 

Preparation

Fill one collection bottle almost to the top (~400ml) with 95% ethanol at the time of deployment. Do not substitute with other kinds of alcohol or other preservatives. Fill out the ‘start date’ on both labels using a PENCIL. As much as possible, use the bottles in sequential order based on the number on their lids. 

Deployment

After arriving at your field site, assemble the trap securely, according to the Malaise trap instruction sheet. When possible, tie the front and/or back ropes to nearby trees for added support. Also, if available, attach the trap poles to a 6- to 8-foot stake or post at its highest points to protect the trap against falling over from high winds, especially if it is placed in an exposed area. For further assembly instructions see the YouTube video on this page. 

Tightly affix the prepared collection bottle to the trap head; tie the white ropes on the trap around the bottle to secure it. Begin the collection on a day of the week you can consistently return to for the duration of the sampling period.

Collection & Monitoring

Collect the catch once a week during the insect activity period (i.e. during non-freezing temperatures). Remove the catch on the same day each week. Use the LIFEPLAN app to scan the QR code on the trap, and then the QR code on the bottle you are about to insert. When collecting the previous sample, wipe the trap head with a clean duster, to avoid dead insects remaining between weeks. Then screw on the new bottle with ethanol for the next sample. Fill out the ‘end date’ on both labels in PENCIL and include relevant comments (examples: trap down at collection, bottle dry, spider web around trap head, etc.). Tear off the internal label and place it inside the bottle. 

Visit the trap frequently if you can to monitor for and repair damage, and to avoid sample overflow. In particular, check the trap after strong winds or heavy rain. In the event of damage, malfunction or other concerns contact lifeplan@helsinki.fi for maintenance suggestions and replacement parts.

Storage

When in the field, store the sample in a shaded cooler, shielded from light. When back from the field, ideally, place the samples in a standard household freezer (i.e. -20°C) for storage. Ensure that the entire insect mass is fully submerged in ethanol before storage; add fresh ethanol to the sample bottle if needed.  

If a freezer is unavailable, store the samples in a refrigerator or cool, dark location. This is critical to preserve the DNA in the samples; improper storage will result in DNA degradation rendering samples unusable for DNA sequencing (e.g. under constant light, heat or variable temperatures). 

Shipment

Just before shipment, decant off most of the ethanol from each bottle using the decanting lid and nitex filters provided. Thoroughly rinse the nitex filter between samples.  

There is a 30ml ethanol maximum per container for shipment and a maximum total of 1L ethanol per package as per International Air Transport Association (IATA) regulations. To follow this protocol, drain the ethanol right down to the insect mass. Shipping 26 samples at a time is recommended. Make sure each bottle is tightly sealed and place them into a plastic bag before packaging in a box.   

Dispatch samples to the Centre of Biodiversity Genomics, Guelph, Canada using cardboard boxes of appropriate size. Contact ccdbcol@uoguelph.ca for further shipment instructions.

Shipment checklist

The first batch of samples should be shipped to CBG after 26 weeks of collecting. Use the checklist below to prepare your outgoing package.

  • Notify ccdbcol@uoguelph.ca, cc: lifeplan@helsinki.fi, that you are ready to ship a package; indicate the number of samples you will include and confirm shipping costs. 
  • Please send a photo of the Malaise trap and its surrounding habitat to lifeplan@helsinki.fi be used for media purposes
  • Determine export requirements from country of origin; obtain all proper permits if needed (Canada does not require import documents for scientific specimens)
  • Prepare the samples for shipment immediately before shipping: decant most of the ethanol from each bottle using the materials provided (see instructions above) and close each bottle tightly to avoid leakage 
  • Place samples in large plastic bag(s) before packaging them into an appropriate sized box
  • Make sure to put a zero (or any permissible minimum) value on the customs declaration form as the samples have no commercial value
  • For the customs declaration, please write 'Insect Specimens for Scientific Study Only (dead), No Commercial Value' 

  • Because the package will contain some ethanol, place the "Scientific Research Specimens" stickers provided on the outside of the package
  • Address the package to the following: 

    Kate Perez - Sample Submission 
    University of Guelph 
    Biodiversity Institute of Ontario 
    50 Stone Road East 
    Guelph, Ontario 
    CANADA 
    N1G 2W1 
    Phone: 519-824-4120 x56393

NOTE

Root sampling will not be part of the core LIFEPLAN sampling, but will instead be a voluntary, shorter module. Soil sampling will be part of the core protocol, but the selection of sampling points will be modified in August 2020.

Equipment provided

  • Disposable gloves
  • Tissue paper
  • Hard 5-cm inner diameter PVC pipe
  • Clear plastic bags
  • Paper bags
  • Airtight Zip-lock bags with QR codes
  • Silica gel

Equipment you provide

  • Knife (sharp and strong, at least 5 cm blade)
  • Gardening spade
  • Scissors
  • Tablespoon
  • Ruler to measure 5 cm depth
  • Ethanol / flame
  • Cooler box: styrofoam or similar
  • Freezer (preferred) or refrigerator for sample storage

Sampling schedule

At each site, you should take two types of soil samples eight times a year: soil samples and samples of roots of known plants. The eight sampling events should be distributed evenly over the entire plant growing season. Thus, in the Arctic region, the sampling times will be distributed along the few months of plant growth (i.e. the snow-free season), whereas in tropical regions the sampling times will be distributed over the whole year. As illustrated in Figure 4 below, for each plant individual, you should collect two Sample Pairs, each pair consisting of a root sample and a soil sample.

Root and soil sampling in Project Lifeplan, University of Helsinki.

Figure 4: Root and soil sampling with different types of plants. A root sample and soil sample from the same plant make a Sample Pair. For each target plant, take two Sample Pairs.

 

Root and soil sampling schedule, Project Lifeplan, University of Helsinki.

Figure 5. Root and soil sampling schedule. Each sampling event consists of 6 Sample Pairs from three different plant species, and 2 Sample Pairs from pooled samples from ten plant species, for a total of 8 Sample Pairs. One Sample Pair = 1 root sample + 1 soil sample.

Focal plant species selection

Fill in the spreadsheet by clicking here

For each location, we will select three focal plant species. The focal plant species should fulfill the following criteria:

1.    The focal plants should be known to establish ECM (ectomycorrhizal) associations. Therefore, predominantly herbaceous families such as Poaceae or Asteraceae are excluded. However, if your study region does not have any plants that form ECM associations, then plants that form other kinds of fungal associations can be selected.

2.    The focal plants should be ones where it is possible to sample their fine roots (under 2 mm in diameter). The feasibility can be best tested by uprooting individuals and examining their root structure. Tree species with deep roots are difficult to sample.

3.    The focal plants should, if possible, be ones that are present at both the urban and the natural site (for sites included in the global design). If there are no shared species between the two sites, different species can be used.

4.    The focal plants should be widespread, so that same species can be shared between as many global locations as possible.

Criterion 4 requires coordination among all the sites. To achieve this, we ask each participating site to provide suggestions of focal plant species that would fulfill criteria 1-3 for their site, and that would additionally have high potential to fulfill criterion 4 by having a wide range. You can suggest as many plant species as you like, preferably at least five. Provide your suggestions hereby finding your site name (listed in the first column), entering your name in the next column and adding scientific names of plant species in the subsequent columns.

Sampling

Each sampling event: take two Sample Pairs (root and soil) of 3 focal plant species, AND two Sample Pairs of pooled root samples and pooled soil samples of 10 different plant species (8 Sample Pairs in total). See Figures 4 and 5.

To avoid contaminating and cross-contaminating the samples, wear disposable gloves and change them each time when handling soil or roots from a different point. Clean the tools with ethanol or flame between the sampling points.

Once you have dug up the plant for the root sample using a knife or spade, you should gently clean the fine roots (under 2 mm diameter) of soil particles by hand (using disposable gloves). Clip the fine roots into a tissue.

Store the replicate root samples separately by wrapping them in tissue paper and placing them in individual paper bags, which are then placed inside individual plastic zip-lock bags containing silica gel, which will draw out the moisture. If the root samples are wet, then it is recommended to change the tissue paper and silica gel within 24h. 

The soil samples are taken using a 5 cm diameter pipe close to (i.e. at a distance of ca. 10 cm from) where the root sample was taken. Press the pipe into the soil to make a mark, and use a knife to cut a 5 cm deep circle using the mark as a guide. Use a tablespoon to dig out the resulting core to a depth of 5 cm into a clear plastic bag. Clear the soil of big particles such as stones, litter, wood pieces and plant roots, picking them out with gloved hands. Then take a subsample of three tablespoons of soil into a paper bag. Place the paper bag inside a zip-lock bag containing silica gel, which will draw out the moisture. Again, store the two replicate soil samples in different paper and plastic bags. 

Store dried samples in a freezer and mail to LIFEPLAN as soon as possible.

In the case of small plants, you should sample a different plant individual every time. In the case of trees, a different individual is also targeted each time. Note that this implies that the focal species selected should be sufficiently abundant, as over the 6 years of the project duration 48 individuals will be sampled per site (6 years x 8 sampling times). If this proves unfeasible, then we will opt for an alternative design.

In addition to the paired root and soil samples, you should collect two pairs of pooled root samples and soil samples (Figure 5). For these, you should sample the roots of 10 plant individuals each representing a different species (randomly selected from those available within the sampling plot), pooling these into a single sample. At the same time, you should sample a soil core next to each of these 10 plant individuals, and mix these cores into a separate sample. The identity of the 10 plant species can vary freely between sampling events, and needs not to be noted. (However, you are encouraged to write them down if you hold sufficient taxonomical expertise). There is no need to keep track of which plant individual has already been sampled for the additional sampling.

In summary, for each sampling event, you should take a total of eight root samples and eight soil samples. During the eight sampling events of each year, you will thus accumulate a total of 64 root samples and 64 soil samples. Samples are shipped to the LIFEPLAN project team as soon as possible.