A new design for feeding the predatory mite, Amblyseius swirskii Athias-Henriot (Acari: Phytoseiidae), on a liquid artificial diet

INTRODUCTION

Amblyseius swirskii Athias-Henriot (Acari: Phytoseiidae) is a generalist phytoseiid mite. It has been used for biological control since 1962 (van Lenteren 2012).

Efficient mass-production methods are required to increase the successful use of biological agents (Blackburn et al. 2016). The mass rearing of predators can be complicated; most are reared on artificial diets, which often require supplementation with honey or sugar solutions (Thompson 1999). Commercial production of A. swirskii based on storage mites needs both space and labour to maintain large cultures of the prey, and may lead to health problems for workers. The accessibility of a suitable artificial diet could eliminate the problems mentioned above (Riahi et al. 2017).

Different artificial diets have been developed for this predator, but using liquid diets complicates mass production of it (Nguyen et al. 2014 a). Nguyen et al. (2015) fed phytoseiid mites on a liquid artificial diet. The artificial diet was absorbed on a small piece of filter paper, but some mites died due to unnatural causes. Nguyen et al. (2014 b)found that A. swirskii had no difficulty handling and feeding on powdered solid artificial diets.Vangansbeke et al. (2014) used dry decapsulated cysts of the brine shrimp, Artemia franciscana (Branchiopoda: Artemiidae) as food supplementation to feed the predatory mite, Amblydromalus limonicus (Acari: Phytoseiidae). San et al. (2020) evaluated the suitability of brown sugar for Carpoglyphus lactis (L.), which was used for rearing A. swirskii. They found that brown sugar resulted in low numbers of predator and prey mites. They attributed that to the relative moisture content and adhesiveness of the brown sugar medium, which inhibited mite movement.

Due to their adhesive nature, semi-liquid diets based on fresh pork liver homogenate generally cause high mortality of young larvae of the predator Harmonia axyridis (Zhang et al., 2008). Sun et al. (2018) observed that after almost one day of exposing the semi-liquid diet to air, a membranous layer was formed on the outside surface of the diet and it looked hard to feed on. To improve the physical properties of that artificial diet, they used the fine powder to develop a mushy diet to replace the semi-liquid diet. They found that the infant larvae had a higher survival rate on a mushy diet than on a semi-liquid diet.

Methods of mass production of biological control agents must be optimized individually (Thompson 1999).

Given the foregoing, feeding predaceous mites, such as A. swerskii, on a liquid artificial diet is often encountered with three obstacles: First, some mite individuals stick to the diet and die. Second, the diet surface stiffens quickly and becomes unsuitable for feeding. Third, there is difficulty getting rid of the stale diet without injuring or losing some individuals. Therefore, the present study was adopted to overcome the above-mentioned obstacles and to facilitate the rearing of that mite for study or for commercial purposes.

MATERIALS AND METHODS

Preparing the main part of all the feeding designs

As shown in Fig. 1, the main part of all the feeding designs was represented by a 13-cm-diameter round plastic dish comprising a circular groove along its margin. To prevent the mites from escaping, the method created by Elshazly (2022 a) was applied. Hereupon, phosphoric acid (3%) was placed in the groove. Moreover, the mites were confined to the central area of the dish (10 cm in diameter), which was surrounded by a five-millimeter-high wall. A 0.5-millimeter-diameter cotton thread was laid on the outer bevel of the wall top. To ensure continuous saturation of the thread, one of its tips was immersed in the groove liquid. The wall was erected by using a hot melt glue stick and a compatible electric gun

Groove

Wall

Thread

Fig. 1. The main part of all the feeding designs

Implementation of the feeding designs

Six feeding designs were tried out. The basis of each design was connected with the method of presenting the diet to the mites. The bases of the feeding designs were as follows:

1. Placing a diet drop directly on the dish

No modifications were made to that design. The surface of the dish was the place to put the diet drop.

2. Placing a diet drop between two glass slides  

As shown in Fig. 2, a microscopic slide was laid on the dish. When a diet drop is placed on the microscope slide and covered with a microscopic coverslip (1.8 cm × 1.8 cm), it spreads between them. The distance between the slide and the coverslip has to be minute.

Fig. 2. The design based on placing the diet between two glass slides

3. Presenting a diet drop via a thread connected to a little of the diet

As shown in Fig. 3, a 20-ml plastic pot was utilized to place the diet stock in. The outer side of the pot was stuck to the dish margin. A 2.5-cm-long tube (3 mm in diameter) was stuck to the dish surface; it penetrated the pot side. On the same straight line as the tube, a similar tube was stuck to the dish surface, leaving a space of eight cm between the two tubes. A hole was made in the side of the pot opposite the tube opening but at a higher level. A 0.5-mm-diameter cotton thread was inserted into the hole and passed straightly through the two tubes. The opposite tips of the two tubes were tightened. A plastic bar was fastened inside the pot to keep the thread near the pot bottom for continuous saturation. The rest of the spool from which the thread was pulled out was kept beside the pot so that the used pieces of the thread could be replaced continually by pulling. The thread located between the two tubes was allocated for mite feeding.

Fig. 3. The design based on presenting a drop of the diet via a thread connected to a little of the diet

4. Placing a diet drop in a tiny plastic pot

As shown in Fig. 4, a plastic pot with a capacity of 0.04 ml was put on the dish to place the diet dropin.

Fig. 4. The design based on placing a drop of the diet in a tiny plastic pot

5. Placing the dish on which a drop of the diet is located, in a tray containing a little water

As shown in Fig. 5, the dish allocated to that design was placed in a tray containing a little water. The water level was below the dish surface. The tray was tightly covered with a glass lid.

Fig. 5. The design based on placing the dish on which a drop of the diet is located, in a tray containing a little water

6. Placing a diet drop on a piece of cloth connected to a water-saturated thread  

As shown in Fig. 6, a piece of cloth (1 cm × 0.7 cm) was laid on the dish; it was the site to put the diet drop on. A 10-cm-long thread was utilized to supply the cloth piece continuously with water. A four-ml vial of water was placed in the dish. One of the thread tips (1 cm long) was laid on the cloth; meanwhile, the other tip was immersed in the water existing in an adjacent vial. The lid hole across which the thread passed was rather loose.

Fig. 6. The design based on placing a drop of the diet on a piece of cloth connected to a water-saturated thread

Replicates

For each feeding design, ten replicates were set up, without taking into consideration the escape-preventing measures, to define the duration of the liquid-phase of the diet. Most of the ten replicates were damaged due to the reduction in the diet amount resulting from examining them. Therefore, only three replicates were considered to determine the duration of the liquid-phase of the diet. It's worth stating that no mites were placed on the dishes allocated for that purpose.

Moreover, for each feeding design, three other replicates were set up, taking into consideration the escape-preventing measures, to determine the numbers of the mites that would stick to the diet.

Obtaining the mite individuals

Individuals of A. swirskii were collected from cotton plant leaves by using a fine brush. For successful pickup, the brush hair was sodden so that the mites could be attached to it easily. The mites were starved for 24 hours before being placed in the dishes. Fifty mite individuals were placed in the dish of each replicate. During the study, individualsof A. swirskii were counted considering the taxonomic identification.

Preparing the diet

A diet composed of skim milk, honey, yolk, baker's yeast, and an amino acid solution of 10% at a volume ratio of 100:1:1:20:1, respectively,was preparedimmediately before introducing it to the mites. According to Elshazly (2022 b), the diet used for this study was found to be the most appealing to A. swirskii when compared with other diet combinations.

Providing the mites with a liquid diet

A diet drop of 0.04 ml was presented to the mites of each replicate. The diet was presented to the mites at night, when most mites move actively. Abd El-Tawab et al. (1982) found that predation by A. swirskii was greater under complete darkness or a short photo-phase (8 h). Diet drops were placed in the appropriate locations for each design. As regards the design based on presenting a diet drop via a thread connected to a little of the diet, two ml of the diet were placed in the pot; meanwhile, the thread got saturated by pulling it. It is worth stating that, in a separate assessment, the diet quantity sufficient for saturating an 8-cm-long thread was found to be 0.04 ml, which is equal to the same diet quantity used for each of the other designs. When the feeding-allocated piece of thread dried and turned solid, the thread was pulled to be replaced with the next saturated piece of thread.

Estimating the duration of the liquid-phase

Since the wet layer of the diet surface is the mite-feeding location, the duration of the liquid-phase of that layer was estimated.The average duration of the liquid-phase was calculated for each design. The values concerning all the designs were arranged in ascending order. Notably, the shiny appearance of the diet surface indicated the liquid phase.The liquid phase was distinguished by touching the diet surface with a brush or a piece of a handkerchief, which absorbs the liquid and turns wet. Rationally, the brush and/or the handkerchief tissue absorb a portion of the diet they touch, which is considered a change in the experiment provisions. That is why ten replicates were set up for that evaluation, despite needing only three.

As regards the design based onpresenting a diet drop via a thread connected to a little of the diet, the duration of the ability of the diet located in the pot to saturate more thread was estimated as well.

Estimating the percentages of the mites that congregate to feed on the diet

The numbers of the congregating mites, in 10, 20, and 30 min to feed on the diet were recorded and the percentage of each category was calculated. For each design, the average percentage of the feeding mites was calculated. The estimated values concerning all the experimental designs were arranged in ascending order.

Estimating the percentages of the mites that adhere to the diet

For each replicate, the mites that stuck to the diet within the first 10, 20, and 30 minutes of the liquid-phase span were counted and the percentage of each category was calculated. For each design, the average percentage of the stuck mites was calculated. The values concerning all the designs were arranged in ascending order.Likewise, for each design, the average percentage of the mites that stuck to the diets within the whole liquid-phase span was calculated. Exceptionally, for both of the designs based on using a tray and using a vial, the mites that stuck to the diet within the first 24 hours were considered because the liquid-phase span of each of the two designs was long-lasting.

Estimating the duration of sucking the diet up

For each feeding design, three replicates were set up to define the duration of sucking the diet. One mite was placed beside the diet to start sucking. As regards the designs in which the diet surface solidifies quickly, more diet was added until the mites were satiated.

Conditions of the experiments

The experiments were conducted at a temperature of 30 ± 5° C, relative humidity of 70 ± 5%, and a 16:8 hr. (L: D) photoperiod.

Statistical analysis

The data were analyzed using ANOVA, with three replicates for each treatment. The least significant differences (L.S.D.) at the 0.05 ≤ level were determined according to the computer program CoStat software and Duncan's Multiple Range. 

RESULTS

Parameters of the liquid-phase span are shown in Table 1. Parameters of the percentages of the mites that congregated to feed on the diet are shown in Table 2. Parameters of the percentages of the mites that adhered to the diet are shown in Table 3. Parameters of the durations of sucking the diet are shown in Table 4. Moreover, there were some remarks on the different designs as follows:

1. Placing a diet drop directly on the dish   

The margin of each drop solidified in 15 minutes.

2. Placing a diet drop between two glass slides

Small bubbles appeared between the two slides and increased with time.

3. Presenting a diet drop via a thread connected to a little of the diet

The diet saturating the thread remained in the liquid phase for 3.2 hours. The liquid diet located in the pot had the ability to saturate more of the thread for up to five hours; then, it coagulated. Accordingly, the total possible feeding span attained from the diet located in the pot would last for eight hours. Mites weren't jeopardized while changing the diet-saturated thread.

4. Placing a diet drop in a tiny plastic pot  

The level of the diet surface was descending with time. The percentage of the evaporated liquid, until turning to the surface-solidification phase, amounted to 75%.

5. Placing the dish on which a drop of the diet is located, in a tray containing a little water 

Over a month, the diet didn't turn solid as long as the humid air was available around it. Dewdrops existed continuously on the inner surface of the tray lid.

6. Placing a diet drop on a piece of cloth connected to a water-saturated thread

Over a month, the diet didn't turn solid as long as the water supply was available.

Table 1: Parameters of the duration of the liquid-phase

Averages followed by the same letter are not significantly different at the P < 0.05 level.

Ranks are arranged in ascending order.

Table 2: Parameters of the percentages of the mites that congregated to feed on the diet    

Averages followed by the same letter in the same column are not significantly different at the P < 0.05 level.

Ranks are arranged in ascending order.

Table 3: Parameters of the percentages of the mites that adhered to the diet

Averages followed by the same letter in the same column are not significantly different at the P < 0.05 level.

Ranks are arranged in ascending order.

* W. F. D.: within the first day

Table 4: Parameters of the durations of sucking the diet

Averages followed by the same letter are not significantly different at the P < 0.05 level.

Ranks are arranged in ascending order.