Preparing eugregarine parasites and their cricket host Acheta domesticus as a model for gregarine infection studies

Abstract

The increasing global demand for house crickets (Acheta domesticus) necessitates effective health measures. Despite their abundance, the effects of gregarines on these hosts remain underexplored. We present a method for controlled gregarine infection and maintenance of gregarine-free cricket populations. This system, adapted from prior work, is essential for experiments on gregarine infection dynamics, providing insights into parasite evolution and host-parasite interactions within the Apicomplexa group. This protocol includes rearing and maintenance of gregarine-free cricket populations for experimental purposes, gregarine production for oocyst solution and cricket infection, and gregarine infection assessment.

Graphical abstract

Specifications table

Subject area:	Immunology and Microbiology
More specific subject area:	Insect parasitology and protistology
Name of your protocol:	Preparing eugregarine parasites and their cricket host Acheta domesticus as a model for gregarine infection studies
Reagents/tools:	Reagents and basic material (further material will be required in specific protocol sections: •  Chicken feed pellets (e.g., Gold 4 gallico, Versele-Laga Belgium) •  Distilled water •  Tap water •  Sand, vermiculite, or coconut fibre for egg laying substrate •  Bug Water Crystals (polyacrylamide) •  25 % and 10 % (w/v) autoclaved sucrose solution • Autoclaved 1x PBS • Ethanol 70 % Tools: • Incubator set at 30°C and 60-70 % relative humidity • Plastic boxes with lids (with a minimum height of 22 cm, e.g. Ikea articles 103.930.64 and 303.930.63) • Driller for ventilation holes in lids • Cardboard egg trays • Electric grinder (e.g., coffee grinder) • Metallic spoon and/or hand-held vacuum cleaner • Plastic lids (8.5 cm diameter) for feed and water gel containers • Plastic petri dishes (9 cm diameter, 1.5 cm height) for egg laying
	• Nylon net (500 µm) for egg laying dishes • Glass petri dishes (9.5 cm diameter) for gregarine manipulation • Dissection Stereomicroscope (16-40x magnification) • Soft forceps • 3 mL Pasteur pipette • 1.5 mL and 2 mL Eppendorf tubes • 50 mL centrifuge tubes • Pipette with 200 µL and 10 µL tips • Micro-centrifuge • 1.5 mL Micro-pestle • Small scissors • Circular saw or hacksaw • Mini bench vice • Wood file • Hot glue pistol and glue sticks •  Hard nylon meshes (500 µm, 1 mm, and 2 mm) •  Tape •  Sieve (250 µm) •  pluriStrainer 100 µm (pluriSelect, Germany: SKU 43-57100-03) •  Funnel pluriSelect (SKU 42-50000-03) •  Connector pluriSelect (SKU 41-50000-03) •  Filter paper •  Scale (1 mg precision) •  Medicinal cup or glass beaker (50 mL) •  Click counter
Experimental design:	We developed a method for controlled gregarine infection and maintenance of gregarine-free crickets using a mesh system inspired by Harry [8]. This system allows for the production of gregarine-free control crickets, enabling group experiments with controlled gregarine infection
Trial registration:	NA
Ethics:	No ethics application are required to work with insect hosts, but we follow the basic animal welfare guidelines as well as the three Rs recommendations: Replacement, Reduction, and Refinement.
Value of the Protocol:	•  No standard protocol and insect model for insect gregarine infection •  Opportunity to better understand the ecological role of gregarines in orthopteran host •  Make advanced research in protistology

Background

The house cricket (Acheta domesticus) is reared worldwide for food, feed and research purposes [10,11]. Crickets are frequently reared on an industrial scale to provide feed to zoos and pet stores. In Europe, A. domesticus is used as an ingredient in sustainable food sources, e.g., in protein bars. Moreover, crickets, especially Gryllus spp. and A. domesticus, are becoming cutting-edge animal models, readily available and cost-effective to acquire and maintain in large numbers [12].

As the production of insects like A. domesticus increases for food, feed, and research purposes, safeguarding their health becomes important in order to maintain thriving cultures. While these insects harbour several parasites, gregarines are particularly abundant [7]. These gut parasites are widespread among insects, including mass-reared species, yet their impact on host fitness and their host range remain largely unexplored. A. domesticus is known to be the typical host of the eugregarine Leidyana gryllorum, [20]. Eugregarines are known for their simple life cycle comprising sexual (gametogony) and asexual (sporogony) phases, producing an environmental stage. The Leidyanidae family, solely composed by the Leidyana genus, primarily includes parasites of lepidopteran and orthopteran hosts [3].

Utilising infection models will offer insights into fundamental questions regarding parasite evolution within the Apicomplexa group, and shed light on the biology and ecological role of gregarines [15]. One significant challenge in working with these parasites is the difficulty of keeping insect cultures parasite free. Previous studies describe instances of ‘heroic treatment’ to remove gregarine oocysts from Tenebrio molitor eggs [17], as well as the use of a ‘pvc isolator system’ with filtered air to rear gregarine-free Schistocerca gregaria locusts [1]. Therefore, we aimed to produce a complete method to assist researchers to implement controlled gregarine infection and to maintain gregarine-free crickets with a mesh system inspired by Harry [8]. This mesh system is mainly designed to obtain gregarine-free control crickets to make it possible to perform experiments with controlled gregarine infection in crickets.

Description of protocol

 

Laboratory cricket rearing

According to Clifford and Woodring [2], the life cycle of A. domesticus from the first instar (also called pinhead) to adult takes 45 days at 30°C. In our system and at 30°C, the female emergence starts around 36 days, while the male emergence starts around 38 days after eclosion (Fig. 1). The adults start mating 2 days after emergence and females start laying eggs 5 days later. The eggs then need 11 days before hatching (Fig. 1). The crickets are reared at a density of c.a. 100 individuals, or 0.15 crickets / cm² or 0.007 crickets / cm³, in large plastic boxes (length 32 x width 21 x height 22 cm). The following rearing procedure is used (adapted from [18]).

Fig. 1.

A. domesticus life cycle at 30°C. D: day(s).

Materials for laboratory cricket rearing

• -
  Incubator set at 30°C and 60-70 % relative humidity (RH).

  • ○
    Higher humidity is beneficial for young instars, but too high a humidity can be fatal to last instars and adults [2]. Therefore, 60-70 % RH is the optimal humidity through the whole life cycle.
• -
  Transparent plastic boxes with lids (such as Box and Lid).

  • ○
    The box height should be at least 22 cm, as the adult crickets can easily jump out of the box otherwise (subtract the height of the egg trays placed inside the box).
• -
  Driller to pierce holes in lids (0.5 mm diameter) for ventilation of rearing boxes

  • ○
    7 rows of holes for every 2 cm
  • ○
    To maintain sufficient numbers of crickets at all time, have four rearing boxes with ventilated lids and two boxes with intact lids for egg incubation (to allow high humidity for egg incubation).
• -Cardboard chicken egg tray (two sections of 2 × 3 eggs tray are recommended), to provide shelter.
• -Chicken feed pellets (such as GOLD 4 GALLICO pellet), ground to powder.
• -Electric grinder (e.g. coffee grinder)
• -Metallic spoon and/or hand-held vacuum cleaner (such as Mini Vacuum)
• -
  Polymer Crystals to make water gel as source of moisture (e.g., Bug Water Crystals)

  • ○
    Five g of crystals is poured in 700 mL of tap water
• -
  Plastic lids with a diameter of 8.5 cm (height of 8 mm) as feed and water gel containers (e.g., lids for 375ml Plastic Pot)

  • ○
    Smaller lids (6.5 cm diameter) can be used for young instars up to one week
• -Plastic petri dishes for egg laying (9 cm diameter)
• -Egg laying substrate: sand (fine sand particles < 250 µm are optimal to separate the eggs), alternatively use vermiculite or coconut fiber.
• -Distilled water and wash bottle.
• -Nylon net (500 µm), elastic band or cable ties for egg laying dishes.

Procedure for cricket rearing

Before starting the rearing procedure assemble materials listed above and rearing boxes available (Fig. 2). Prepare the cricket feed by finely grinding the chicken pellets into fine powder (this can be sieved through a 500 µm mesh to discard larger particles).

Fig. 2.

Typical rearing box with perforated lid. Box dimensions: 32 × 21 × 22 cm.

Rearing tasks to perform every 3-4 days

• •
  Cleaning rearing boxes (especially for adult crickets)

  • ○
    Remove the faecal matter, dead crickets from the rearing boxes with a spoon or mini vacuum cleaner.
  • ○
    When collecting the faeces for gametocyst isolation (see later procedure 2.), first remove any cadavers and then collect the faeces.
• •
  Replace the chicken feed in a new clean plastic lid

  • ○
    Change of feed can be done weekly depending on consumption and the state of the feed (discard if wet and mouldy).
• •
  Replace water gel container

  • ○
    The water gel can be re-moisturised with a wash bottle supplied with tap water (add directly water to the water crystals with the wash bottle). If the water gel is mouldy, replace it entirely.
  • ○
    Slices of carrot can be alternative source of moisture, but they usually get mouldy in 2-3 days. Other water sources can be an agarose gel, or a cotton-plugged tube.

Rearing tasks to perform every 7-14 days

• •Clean climate chamber/incubator with a sponge cloth and soapy water
• •Refill the water tanks in incubator to maintain humidity
• •Replace cardboard egg trays if moist or chewed up

Egg laying (c.a. 47 days after hatching)

• •
  Seven days after adult emergence, add the base of a petri dish with the moist egg-laying medium (e.g., sand) and allow the females to oviposit for 24 h. This time is sufficient to harvest plenty of eggs.

  • ○
    Make sure to cover the petri dish with a net and secure it tightly using an elastic band or cable tie to prevent the crickets from burrowing into the medium (Fig. 3).
• •
  Transfer the petri dish with eggs to a new rearing box fitted with an intact lid without holes to maintain high humidity. Eggs can easily become too dry and will not hatch.

  • ○
    Note the egg laying date and later the hatching date.
• •Once the pinheads hatch, transfer ca. 0.07 g (corresponding to approx. 100 individuals) to a new rearing box (with a ventilated lid) containing cardboard egg trays, feed and water gel.
• •At the end of the crickets’ life cycle, place the rearing box in a cold chamber (4-5°C) for 30 min. Then, crickets and egg trays can be transferred to freezer bags, which are sealed and frozen at -20°C. Finally, clean the emptied rearing boxes in a dishwasher.

Fig. 3.

Female cricket (Gryllus bimaculatus) laying eggs on wet sand. Note that A. domesticus lay eggs in the same way. The nylon net's mesh used to protect the egg laying medium from crickets digging is 500 µm.

Gregarine isolation and preparation of a solution of purified oocysts

The aim of this procedure is to harvest gametocysts from cricket faeces and induce the release of oocysts for experimental studies. The oocysts solutions can be used for cricket infection or DNA/RNA extraction. For more clarity, the Fig. 4 represents the life cycle of the gregarine infecting A. domesticus (Leidyana gryllorum). The life cycle is usually completed in 4-5 days at 30°C.

Fig. 4.

Leidyana gryllorum life cycle. (A) Oocysts represent the infectious stage of gregarines. The oocysts are ingested orally by the host and will release sporozoites (8). Sporozoites then infect the host's intestinal cells and develop to trophozoites. (B), (C) Intestinal stages of the gregarine with a young trophozoite in (B) and an older individual or gamont in (C). (ep) Represents the trophozoite's epimerite that allows anchoring to the host cell. (D) Gametocyst, usually observed in the cricket hindgut. Gametocyst is the stage allowing the gregarine sexual reproduction and results from the association of two gamonts, which is called syzygy. Syzygy of L. gryllorum gamonts is rarely seen as happening late in its life cycle. The formed gametocyst is then extruded in the environment with the faeces. (sp) Represents developing sporoducts, from which oocyst chains will be later extruded. (E) Isolated gametocysts placed on a black filter paper starting their dehiscence (i.e. the release of oocyst chains); the oocyst chains are barely visible as very thin white threads. Asexual reproduction occurs in the oocysts resulting in sporozoites.

Materials

• •Dissection Stereomicroscope (recommended 16-40x magnification)
• •Nymphs or adult crickets with gregarine infections
• •Metal spoon or mini vacuum for faeces collection
• •Glass petri dishes (9.5 cm diameter) are recommended for gregarine manipulation as they can adhere to plastic surfaces
• •Freshly collected cricket faeces with gametocysts
• •Soft forceps
• •Autoclaved 1x PBS or 1x Ringer's solution
• •25 % and 10 % (w/v, distilled water) autoclaved sucrose solution
• •3 mL Pasteur pipette
• •2 mL Eppendorf tubes
• •50 mL centrifuge tubes
• •Optional: 100 µm pluriStrainer® (pluriSelect, Germany)
• •Pipette with 200 µL tips
• •Micro-centrifuge for 1.5 and 2 mL Eppendorf tubes
• •Micro-pestle (for 1.5 mL Eppendorf tube)
• •Small scissors
• •Incubator at 30°C with water trays to maintain 70 % RH

Procedure

• 1. Collect cricket faeces from rearing boxes using a metal spoon. Gently incline and tap the rearing box to concentrate the faeces in one corner (Fig. 5) and transfer them to a Petri dish. It is preferable to discard the faeces from the rearing box the day before to ensure fresh faeces for collection and extrusion of gametocysts. It is best to collect the faeces from at least 14 days old cricket nymphs which produce larger faecal pellets than younger stages making them easier to observe.

• 2. Examine the faeces under a dissection stereomicroscope, where gametocysts measuring an average of 150 µm can be observed at a magnification of 16x. Pick and transfer the faecal pellets containing gametocysts with soft forceps to a 2 mL Eppendorf tube containing 1 mL of 1x PBS solution. Approximately 200 gametocysts are sufficient for harvesting enough oocysts for infection experiments, but more is better. The collection process typically takes 30 to 60 min, depending on gregarine prevalence.

• 3. Incubate the resuspended faeces and gametocysts in the 1x PBS solution at room temperature for 1 h to release the gametocysts from the faeces. The process can be facilitated by gently pipetting up and down using a 3 mL Pasteur pipette.

• 4. Once gametocysts are released from the faeces, perform a sucrose gradient separation [13].

• a.Add 20 mL of 25 % sucrose solution to a 50 mL centrifuge tube, then gently add a 20 mL layer of 10 % sucrose solution on top while the tube is held at a 45° angle, this allows to gently pour the 10 % solution and form the gradient (Fig. 6). Without maintaining a soft angle when pouring the 10 % layer, there is a risk of mixing the two solutions.
• b.Allow the solution to stand up right at room temperature for at least one hour, and a band of gametocysts should appear at the interface of the two sucrose solutions (Fig. 7A).

• 5. Optional: The gametocysts band is extracted with a 3 mL Pasteur pipette and placed onto a 100 µm strainer to remove the sucrose solution (Fig. 7B). Afterwards, the strainer is inverted, and the gametocysts are rinsed off with 1x PBS using a Pasteur pipette into a glass petri dish containing 1x PBS.

• 6. The gametocyst band within the sucrose solution is then transferred from the 50 mL tube (or washed off the mesh from step 5) to a glass petri dish containing 1x PBS to be rinsed. Glass petri dishes are preferred as the gametocysts can adhere to plastic surfaces.

• 7. Gametocysts are transferred to a new glass petri dish containing 1x PBS using a pipette with a 200 µL tip. This tip size is selected based on the size of the gametocysts, facilitating the transfer of many gametocysts without disturbing them. Tip: gametocysts can be gathered by gentle agitation of the Petri dish by hand. This will concentrate the gametocysts in the centre (see the video attached here: Gametocysts_agitation.mov).

• 8. Afterwards, the gametocysts are transferred again with a 200 µL pipette tip to a 1.5 mL Eppendorf tube, while pipetting as little as PBS solution as possible.

• 9. Centrifuge the tube with a micro-centrifuge for 30s. Ensure equal reciprocal distribution of tubes for balancing the centrifuge.

• 10. Remove as much PBS as possible by first using a 200 µL pipette tip, and then with a 10 µL pipette tip (Fig. 7C). Tip: to remove the last bit of PBS, press the 10 µL pipette tip firmly against the bottom of the Eppendorf tube to draw up liquid without picking up any gametocysts. The small space left will avoid pipetting gametocysts.

• 11. Finally, a hole is made in the lid of the Eppendorf tube with the tip of small scissors for ventilation, as too high a humidity can be lethal for the gametocysts [6]. Then, the gametocysts are incubated at 30°C in the tube, for at least 4 h to let them mature and produce oocysts. The incubation can preferably be done overnight.

• 12. Once the gametocysts have matured and started to dehisce, 500 µL of 1x PBS are added to the tube (Fig. 7D). Tip: if possible, processing mature gametocysts that have not yet dehisced (i.e. presence of sporoducts) is preferable for oocyst collection, as the oocysts will then be concentrated within the gametocysts, rather than being released as oocyst chains into the air of the tube, where they can adhere to the tube's wall.

• 13. The gametocysts in the tube are pelleted with a micro-centrifuge for 30s and crushed manually with a micro-pestle. This step is repeated three times. Make sure to agitate the micro-pestle into the solution to avoid removing oocysts.

• 14. Draw a 20 µL volume of the solution by pipetting up and down few times, and load this volume into a Fuchs-Rosenthal haemocytometer. Count 5 large squares (1 mm²) to estimate the oocyst concentration.

• 15. For counting oocysts, use a light microscope at 200x magnification (Fig. 8). Eventually, use the following formula to estimate the concentration of the solution:

  $$C=\frac{Oocystscounted}{Areascounted\left[5\times 1.0m{m}^2\right]\times Chamberheight\left[0.2\mu l\right]}=Oocystscount/\mu l$$

• 16. The solution is now ready for experimental infection or various nucleic acid extractions.

Fig. 5.

(A) Faeces collection from cricket rearing box. (B) Observation of the collected faeces under a stereomicroscope.

Fig. 6.

Making the discontinuous sucrose gradient with an angled 50 mL centrifuge tube. The first layer of 25 % sucrose is poured without precautions but the second layer of 10 % sucrose has to be applied gently and with an angle to avoid any mixing of the two layers.

Fig. 7.

(A) Sucrose density gradient with gametocysts band (red rectangle). (B) Gametocysts distributed on a 100 µm pluriStrainer® mesh (16 x). (C) Isolated gametocysts before and (D) after incubation and early dehiscence, some oocyst chains are visible and sticking to the tube's wall (40 x). It is preferable to process gametocysts before their full dehiscence, as the recovery of the released oocyst chains (sticking to the tube's wall) is more challenging than disrupting cysts full of oocysts.

Fig. 8.

Oocysts (dolioform) counting in one of the small squares (250 × 250 µm) of a Fuchs-Rosenthal chamber. Some of the oocysts are indicated by red arrows. This square has c.a. 50 oocysts.

Creating uninfected control population of A. domestica

Rearing boxes with a mesh in the bottom will allow to rear the crickets free from gregarine exposures (adapted from [8]); a circular saw or a hacksaw; hard nylon meshes of 0.5, 1 and 2 mm; a glue pistol and a mini bench vice is needed.

Making mesh boxes for infection free control host population

Materials

• •Rearing plastic boxes (min. height: 22 cm) with ventilated lids
• •Marker
• •Circular saw or hacksaw
• •Mini bench vice
• •Wood grater
• •Pistol glue and glue sticks
• •Hard nylon meshes of three mesh sizes (500 µm, 1 and 2 mm)
• •Tape

Procedure

• 1.Measure where to cut off the box bottom. A box with a height of 22 cm was for example cut at 5 cm from the bottom. This 5 cm should be the minimum height as oocyst chains from dehisced gametocysts can otherwise reach above the cut bottom.
• 2.Use a marker to draw the cutting line and cut the box with a circular saw or hacksaw while holding it tightly with a mini bench vice against the working bench. Ensure to cover bare skin when cutting the plastic box to protect against hot plastic particles. A wood grater file can be used to straighten and polish the cut plastic edges.
• 3.Apply glue to the edges of the upper box and affix the mesh. Ensure to seal any small holes between the mesh and the box to prevent small crickets from escaping.
• 4.Eventually, use tape to fix the bottom and upper parts of the box together (Fig. 9)
• 5.The boxes can be washed by hand with water and soap; excessive heat (in dishwasher) may loosen the glue and mesh. Regularly inspect for potential holes and seal them with glue.
• 6.Ensure an adequate supply of boxes with different mesh sizes (see below) according to your needs. Tip: If there are insufficient cut boxes, a mesh can be removed under hot water and replaced with a new one, to adapt the size needed rather than making a new box.

Fig. 9.

Mesh system to control gregarine infection. Cricket faeces will fall through the mesh to the bottom compartment, avoiding contact between crickets and gregarine oocysts.

Rearing of control cricket populations

Materials

• •Cricket eggs (7-9 days old)
• •Strainer (250 µm)
• •Hard paintbrush
• •Distilled water
• •Ethanol 70 %
• •Filter paper
• •Container for egg incubation
• •Container with modified bottom (500 µm mesh, for pinheads)
• •Boxes with larger mesh sizes (1 mm and 2 mm, for larger cricket stages)
• •Cardboard egg trays
• •Water gel
• •Microwaved grounded chicken feed
• •Incubators
• •Separate handling equipment and incubation rooms for control and treated units during experiments to avoid contamination of control populations
• •Lab coat

Procedure

• 1. Preferably use cricket eggs that are 7-9 days old, as they are more resilient with a thicker chorion, compared to younger eggs ([14], E.B. pers. obs.).

• 2. Separate the eggs from the sand (egg-laying medium) by washing the sand and eggs on top of a 250 µm strainer using distilled water. Discard the sand after washing (Fig. 10).

• 3. Thoroughly wash the cricket eggs with 70 % ethanol [9], followed by a final rinse with distilled water to remove any ethanol residue.

• 4. Transfer the washed eggs to a wet filter paper using a hard paintbrush, and place them in a sealed container, to ensure high humidity (c.a. 100 % R.H.). Incubate at 30°C until hatching (expect approximately 11 days from egg laying).

• 5. After hatching, move the pinheads to a box with a modified bottom featuring a 500 µm mesh to facilitate faeces removal (see previous procedure 3.1).

• 6. As the crickets grow, transfer them to boxes with larger mesh sizes (1 mm after a week and 2 mm for the late stages and adults).

• 7. Minimise potential contact with oocysts resulting from gametocyst dehiscence by discarding faeces from the box bottom and replacing egg trays, water gel and feed on the mesh floor daily.

• a.It is also recommended to microwave the grounded feed stock for 60s to avoid contamination.

• 8. Always separate the control populations in another room from the normal rearing population to prevent reinfection with gregarines through contaminated material (unwashed equipment, rearing boxes, etc.).

• 9. During experiments, place the control and treated units in separate incubators and handle the units in different rooms. Begin always with the control group.

Fig. 10.

(A) Washed A. domesticus eggs on top of a 250 µm strainer. (B) Zoom on developed A. domesticus eggs, embryos’ eyes can be seen in late egg development.

Gregarine experimental infections

Materials

• •Fresh quantified oocyst solution
• •Plastic lids and water gel
• •Cricket nymphs from control population
• •Metal spatula or small spoon
• •Pipette with 200 or 1000 µL tips
• •Scale (1 mg precision)
• •Medicinal cup or glass beaker (50 mL)
• •Individual container and parafilm for individual infection
• •2 mL Eppendorf without caps and cotton (for individual infection)

Procedure

• 1. Ensure a fresh oocyst solution is available. Note that storing the oocyst solution in the fridge overnight for infection purposes is acceptable, but the viability of the oocysts under these conditions has not been tested. A method for oocyst viability assessment is discussed in the limitation section, but oocyst excystation proved to be complicated and was not explored further. Infection success was indicated as presence of gametocysts in faeces of exposed crickets, which usually occurs after c.a. 4 days of infection for crickets kept at 30°C (see Gregarine quantification for experiment purpose section). Using this approach, experimental infections were always successfully demonstrated with the use of fresh oocyst solutions.

• 2. Add the oocyst solution directly to a known mass of water gel in a cup or beaker, and mix thoroughly with a spatula or spoon to homogenise the gel (see previous method on how to prepare an oocyst solution).

• 3. Distribute the contaminated water gel to the crickets for infection (Fig. 11). Therefore, the experimental group of crickets is exposed to a known concentration of oocysts per gram of water gel (oocysts / g). This approach is suitable for group experiments.

• 4. Optional: perform individual infection by distributing a drop (c.a. 50 µL) of oocyst solution on top of parafilm directly to individualised crickets in 375 mL pots with pierced lids. It can be observed if the cricket consumes the drop. Therefore, the crickets are exposed to a known dose of oocysts compared to the gel exposure. However, it is important to keep solution homogeneous by agitation, and it is recommended to count oocysts in 5-6 drops to ensure expected number and assess variation. Keep the crickets individually and supply them with feed and water by filling 2 mL tubes without caps (plug the tube containing water with cotton) (Fig. 11C).

• 5. To enhance water gel or droplet consumption by the cricket, deprive them of a water source for 24-48 h prior to exposure.

Fig. 11.

(A) Cricket ingesting oocyst contaminated water gel. (B) Individual cricket drinking oocyst drop placed on parafilm. (C) Individual set up.

Gregarine quantification for experiment purpose

To evaluate the efficacy of the artificial infection and the non-infection of the control group, the presence of gametocysts in the faeces can be monitored daily and quantified by the following procedure. In this process, all the faeces collected from each experimental unit can be examined for the presence (qualitative) and number (quantitative) of gametocysts.

Materials

• •50 mL centrifuge tubes
• •Weighting scale (1 mg precision)
• •1x PBS
• •Sucrose (25 % and 10 % solutions)
• •Distilled water
• •Dissection Stereomicroscope (16-40x magnification)
• •3 mL Pasteur pipette
• •300 µm nylon mesh
• •100 µm pluriStrainer® (pluriSelect, Germany)
• •Connector from pluriSelect (pluriSelect, Germany)
• •Funnel from pluriSelect (pluriSelect, Germany)
• •Click counter

Note: material from pluriSelect should not been thrown away (as rather expensive) and can be thoroughly washed with water and soap or with an ultrasonic cleaner/bath if available to reuse the pieces.

Procedure

• 1. Collect all faeces from the bottom of the box and transfer to a 50 mL centrifuge tube. Use a funnel if necessary to guide the faeces into the tube. Before weighing the faeces on the scale, remember to tare the empty tube.

• a.Some feed debris might also fall into the bottom of the box. Gently tap the bottom at an angle so that the faeces are cornered while the lighter feed particles remain in their initial position (Fig. 12).

• 2. Suspend the faeces in 6 mL of 1x PBS overnight, or until the faeces layer is well covered by the PBS, to free the gametocysts from the faeces.

• 3. The following day, layer the faeces suspension on top of a sucrose gradient prepared in a 50 mL tube consisting of 20 mL of 25 % sucrose and 20 mL of 10 % sucrose (w/v, distilled water). Allow the samples to sit for several hours at room temperature to allow the gametocysts to form a band between the two sucrose solutions (Fig. 13A).

• a.The gradient from the oocyst production (Procedure 2.2, Fig. 7A) is clearer since only faeces containing gametocysts are selected, resulting in a reduced faecal material, compared to the current experiment monitoring.

• 4. Once the gradient is formed, extract approximately 20 mL of the suspension around the expected band using a Pasteur pipette. Filter the collected solution through a 300 µm mesh positioned atop a 50 mL tube, where the mesh is secured by using a pluriSelect funnel (Fig. 13B). This allows to remove faecal debris.

• 5. From the previous filtrate, layer the gametocysts, which measure between 100-150 µm, on top of the pluriStrainer® 100 µm mesh and count them under a stereomicroscope at a magnification of 16x - 25x (Fig. 13C).

Fig. 12.

Collecting faeces from the bottom of a mesh experimental unit.

Fig. 13.

(A) Faeces samples into sucrose gradient for gametocysts monitoring. (B) System to filtrate and layer the gametocysts from the gradient. (C) Gametocysts layered on top of a 100 µm PluriStrainer®.

Protocol validation

 

Limitations

In vitro excystation assay, future prospects

The current protocol does not assess directly the oocyst viability prior infection, as briefly stated in the gregarine experimental infection. A way to determine oocyst viability would be to implement oocyst excystation assays. Previous studies suggest that oocysts excyst upon contact with host digestive fluids [5,16]. Briefly, host gut parts are extracted from anesthetised insect hosts, bead beaten in Ringer's solution, and cleared of debris by centrifugation. The resulting extract is filtered through a 0.45 µm syringe filter. In the in vitro excystation assay, the extract is combined with approximately 1000 oocysts from a fresh solution. The wet mount is sealed and observed under phase microscopy for evidence of sporozoite activation and excystation, with observations made at 5 min intervals for up to 1 h or until excystation is evident [5]. Clopton and Gold [4] demonstrated that host intestinal pH can influence the oocyst excystation response, with different gregarine species exhibiting varying excystation rates and speeds at different pH levels. Notably, gut parts of A. domesticus display varying pH levels, ranging from 4.5 (crop) to 7.4 (ventriculus) [19]. During the current work, few trials were made with whole gut parts of A. domesticus and no obvious excystation was observed, perhaps indicating that the pH of the extract was inadequate. The current protocol is sufficient to assess the infection success by recovering of gametocysts from the collected faeces.

CRediT authorship contribution statement

Edouard Bessette: Conceptualization, Methodology, Investigation, Visualization, Writing – original draft, Project administration. Bryony A.P. Williams: Supervision, Validation, Writing – review & editing, Funding acquisition. Nicolai V. Meyling: Supervision, Validation, Writing – review & editing, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

• Data will be made available on request.