Abstract
The effect of Cyrtophora citricola cues on the foraging behaviour of Acrida cinerea was studied by using Ocimum basilicum. The samples of the spiders and the grasshoppers were collected from Sargodha, Pakistan. Thirty leaves of the plants (fifteen were included in the control group and the other fifteen had cues of the spider) were selected both in the single-leaf laboratory experiment and single-leaf plant experiment. The leaves were plucked from the plants in the single-leaf laboratory experiment while in the other experiments, the leaves were attached to the plants. The cues were obtained by moving the spider on the leaves. The size of the leaves was measured before and after the experiment by using leaf grids. The grasshoppers were placed in separate plastic cups along with the leaves and after 24 hours, the percentage herbivory was calculated. In the whole plant experiment, sixty leaves from five plants (twelve leaves from each plant) were selected and marked with paper tape. There were six leaves included in the control group and the other six were cue treated from each plant. The plants were enclosed in translucent porous plastic bags and after three days the grasshoppers were removed and the percentage herbivory was calculated by measuring the size of the leaves. In all experiments, the craving level of grasshoppers was equalized before the beginning of the experiments. The results showed that there was less herbivory in the cue-treated group as compared to the control group and there was a significant difference among them. In the cues effectiveness experiment, three different insect species i.e. bush cricket, house cricket, and house fly were compared with grasshopper. The cues were deposited on the four filter papers in the petri plates by moving the spider on it for three days. Half cue deposited filter paper and half clean filter paper were joined and the specimens were allowed to reside in both areas. The results showed that there was a non-significant difference among them. However, all the species spent more time on the clean filter paper which concluded that the insect pests avoid the cues of the spider.
INTRODUCTION
In the terrestrial ecosystem, spiders are considered as the significant predator of insect pests. In the commendatory environment, the number of spiders increases by almost 1000 individuals/m2. Hence, they play a crucial part in the regulation of the number of the pests in the agroecosystem as they prevent the prey to harm the plants by acting as a presiding predator (Shurin et al., 2002; Tahir et al., 2019). They belong to one of the most diverse classes of the terrestrial Arthropods. As a predator, the spider not only controls the growth rate of the prey by killing them directly and eating them but also has some other indirect effects which cause either morphological or behavioral changes in the prey. Thus, these indirect effects of the spiders are known as non-consumptive effects which are not fatal (Abrams, 2007; Bucher et al., 2015).
As carnivores, they feed on the insects which belong to the various taxa. Besides this, the eggs, the larvae of the insect, and other arthropods are also included in their food (Sharma, 2014). The spiders use different types of cues and these cues are either direct or indirect. The spiders use chemicals like pheromones and waste materials as the direct cues for the predators. The speedily moving prey can get an advantage of it and can ascertain the presence of the predator. Silk and eggs are also included in indirect cues. However, the indirect cues consist of the kairomones obtained from the dead organisms (Kortet & Hedrick, 2004; Persons & Rypstra, 2000; Persons et al., 2001; Ripple et al., 2016). The anti-predatory strategies of the prey rely on the identification of these cues therefore, these cues play a significant role in the interactivity between them (Barnes et al., 2002; Dicke & Grostal, 2001; Persons et al., 2001).
In the terrestrial food webs, the predators have an indirect effect on the plenty of the plant biomass by reducing the number of prey under the process of the trophic cascade. Hence the activity of the upper predators affects the lower ones and eventually, this effect also reaches the primary productivity level. So, when the spiders are present in a particular agroecosystem then the pests try to avoid them and forage at a lower rate. Hence, the yield of the plant rises indirectly, and spider works as the biological agents in the agricultural ecosystems (Estes et al., 2011; King & Hardy, 2013; Schmitz & Sokol-Hessener, 2002; Schmitz & Suttle, 2001; Schmitz et al., 2000; Schmitz et al., 2004; Shurin et al., 2002; Tahir et al., 2009).
Many scientists are now working on different non-chemical methods to control the insect pest in agricultural crops (Crowder et al., 2010; Huvenne & Smagghe, 2010; Klapwijk et al., 2016). The cues of the spiders including their silk are potentially so strong they can significantly reduce the pest attack (Bucher et al., 2015; Rypstra & Buddle, 2013; Tahir et al., 2019).
For this study, the spider, Cyrtophora citricola, was used for the cues’ source as they were very common in the bushes and on citrus. To check the herbivoury, the Chinese grasshoppers (Acrida cinerea) were selected as in the fields of the corn and sugarcane they were present in a large number in the summer season and they fed on the green leaves. Basil plants (Ocimum basilicum) were used in the food for aromatic purposes and were easily available in the market and gardens so to evaluate the foraging behaviour of grasshoppers, they were used. In Pakistan, no study was reported on these species so there was a need to investigate them.
The objectives of this research work were:
- To study the foraging and avoiding behaviour of the Chinese grasshopper.
- To evaluate the roles of spider cues in reducing insect pest attacks
MATERIALS AND METHODS
Cues’ source
The colonial tent web orb-weaver spider (Cyrtophora citricola) was used for cues’ deposition. These spider species were collected from the bushes of Sillanwali (31°49’34.00” N 72°32’23.42” E), district Sargodha, Pakistan during August-September, 2021, and were collected by utilizing the hand-picking method and maintained their temperature at 35-40 ℃. They were placed separately in the falcon tubes (30×115 mm) and at the bottom of the tubes, the sand and clay were placed. These spiders were fed on the fruit fly (Drosophila melanogaster). The tubes were covered with mesh cloth for ventilation.
Selected Insect
The Chinese grasshoppers also known as oriental longheaded grasshoppers (Acrida cinerea) were selected to examine the herbivory of the plant. These insects were collected with the help of the sweep nets from the corn, citrus, and millet field of Sillanwali (31°49’34.00” N 72°32’23.42” E). The insects were kept at 35-40 ℃ in translucent plastic cups (9 cm× 5.5 cm) having wet cotton at the bottom to maintain 60-70% humidity and covered with perforated lids for ventilation. A period of light and dark at 10:14 hours was given to them (Harris et al., 2015). They were given different green leaves and petals of corn, millet, and other grasses. Before the initiation of the experiments, the grasshoppers have fully gratified with the food and after this system the craving level, they were famished for one day.
Selection of plant
Sweet basil plants (Ocimum basilicum) were selected to examine the herbivoury as the grasshoppers fed on its leaves and harmed them. The herbivoury caused by the Chinese grasshopper was determined by its leaves. The plants were taken from the nearby nursery which was present in front of the University of Sargodha (32°4’56.8776” N 72°40’8.8608” E). There was no exposure of any chemical spray either pesticide or insecticide on the plants. All the plants were in fresh condition. There were almost 30-40 leaves on each plant.
Single leaf laboratory experiment
Thirty leaves were selected from different plants and the size of all the leaves was quantified by using leaf grids at the beginning of the experiment. The cues on the fifteen leaves were obtained from moving the spider on it for two days when they are attached to the plant. The leaves were plucked after cue deposition and a small piece of wet cotton was folded around each stalk of the leaves to keep them fresh.
The other fifteen leaves of the control group remained free of cues and were placed in separate plastic glasses having dimensions of 9 cm × 5.5 cm. All the grasshoppers were fully slaked with the food (i.e., leaves) and then remained hungry for 24 hours to keep the craving level the same before the start of the experiment. Then, they all were placed in separate disposable plastic glasses by maintaining an appropriate temperature and humidity level. In each glass, one grasshopper and a leaf were placed to check the herbivoury, and glasses were covered with the perforated plastic lid for 24 hours. After this, both the cue deposited and the normal leaves were gathered from the plastic glasses, and the percentage of the herbivoury convicted by the grasshopper was calculated by using the leaf grids hence, the size of the leaves was measured again at the end of the experiment.
Single leaf plant experiment
From fifteen plants, thirty leaves (two leaves from each plant) were selected for this experiment. These selected leaves were attached to the plants during the experiment and were not pulled off from the plants. The size of all the selected leaves was measured by using the leaf grids before the initiation of the experiment. The cues of the spider, Cyrtophora citricola, for the experimental group were obtained by moving it on the selected fifteen leaves for two days. The remaining fifteen leaves of the control group were without spider cues. All the selected leaves were placed in separate disposable plastic glasses in that position where they remained connected to the plants. All the glasses were covered with perforated lids. All the grasshoppers were fully fed with food before the initiation of the experiment and after it, they remained hungry for twenty-four hours to maintain their hunger level. The plastic glasses were removed after twenty-four hours and the size of all the selected leaves was calculated by using leaf grids to scrutinize the herbivoury of the plants.
Whole plant experiment
Selected five plants were enfolded separately in plastic bags which were translucent and perforated. Twelve leaves were selected from each of five plants, among which six leaves were considered for the experimental group by having spider cues and the other six were in the control group. The leaves were almost similar in size. These selected leaves were characterized by wrapping the paper tape around the stalk of the leaves and the size of all of them was measured. The spiders were left on the leaves for two days to obtain the cues and after this, they were removed. The leaves in the control group were free of cues. There were placed five grasshoppers in each plastic bag and left for three days. After three days, the plastic bags with the grasshoppers were pulled out, and to evaluate the herbivoury of the leaves, the size of all the selected leaves was measured.
Cue effectiveness
Insects like Chinese grasshoppers, Acrida cinera, bush cricket, Hexacentrus japonicus, housefly, Musca domestica, house cricket, Acheta domestica were chosen to check the effectiveness of the cues of the spider, Cyrtophora citricola, and their behaviour towards the cues were evaluated. In the four Petri plates of the size of 100 mm × 15 mm, four filter papers were kept in. A spider was released on the filter paper for three days for the deposition of cues. The Petri plate was wrapped with the pierced shield of plastic. The spider was removed after three days and the filter paper was divided into two uniform pieces. A normal cue-less round filter paper was also divided into two uniform pieces. Half part of the cue deposited filter paper and half of the normal cue less filter paper was fastened cautiously with the aid of the paper tape. The new unite filter paper was kept in the Petri plate and the insects were allowed to reside on the plate for half an hour. Their behaviour was evaluated by seeing their time spend either on the cue-bearing filter paper or the no cue-bearing filter paper with the help of the stopwatch. With one insect and the same plate, the procedure was repeated five times. For all the insects, this same procedure was used to check the effectiveness of the cues separately. For one species, three specimens were used to check the cue effectiveness.
Statistical analysis
The independent t-test was applied to calculate the percentage herbivoury of grasshopper among the control and cue treated groups of all the experiments and one way ANOVA was applied to compare the cue’s effectiveness on the four different species like the Chinese grasshopper, bush-cricket, house cricket, and housefly with the help of GraphPad Prism 8.0.1.
RESULTS
Single leaf laboratory experiment
The Single leaf laboratory experiment was performed to calculate the percentage herbivory of the grasshopper (Acrida cinerea) with or without the cue treated leaves of the plant (Ocimum basilicum) by detaching the leaves from the plant and putting it into the separate translucent plastic cups which were covered with the pierced lid. Among thirty selected leaves, on fifteen leaves the cues of the spider (Cyrtophora citricola) were present while the others were untreated. The percentage herbivory was calculated after 24 h by measuring the size of the leaves. The results showed that the percentage herbivoury of the experimental (cue treated) group was less (3.233 ± 1.212) as compared to the control group (19.47 ± 5.274). The results between both these groups had statistically significant difference (t = 3.000; p = 0.0056; Fig.1).
Figure 1: Percentage herbivoury of the Chinese grasshopper (Acrida cinerea) among the experimental (Cyrtophora citricola’s cues treated) and control group of the Basil plant (Ocimum basilicum) in the single leaf laboratory experiment.
Note: two stars (**) show significant differences among both groups.
Single leaf plant experiment
The Single leaf plant experiment was performed to calculate the percentage herbivory of the grasshopper (Acrida cinerea) with or without the cue treated leaves of the plant (Ocimum basilicum) while the leaves did not pluck from the plant. The grasshoppers which had alike starvation levels were put into the translucent cups of plastic covered with the pierced lid and the glasses were positioned in such a way that the leaves remain connected to the plants. Among thirty selected leaves, fifteen leaves had the cues of the spider (Cyrtophora citricola) while the others were untreated. The percentage herbivory was calculated after 24 h by measuring the size of the leaves. The percentage herbivoury of the experimental group (cue treated) was less (1.360 ± 0.536) as compared to the control group (22.60 ± 7.161). The results of the two groups were significantly different (t =2.958; p = 0.0062, Fig. 2).
Figure 2: The percentage of herbivoury by Chinese grasshopper (A. cinerea) among the experimental (C. citricola’s cues treated) and control group on the basil plant (O. basilicum) in single leaf plant experiment.
Note: two stars (**) show significant differences among both groups.
Whole plant experiment
In this experiment, the percentage of herbivoury of the grasshopper (Acrida cinerea) was calculated by the five basil plants. Each of five plants, six leaves had cues of, Cyrtophora citricola, and the other six were untreated having no cues. All the plants were enclosed in porous translucent plastic bags along with the grasshoppers. All the grasshoppers had the same hunger level. There were five grasshoppers in each bag. After three days, the percentage of herbivoury was calculated which was far less in the experimental (cue treated) group (0.4933 ± 0.1797) than in the control group (26.57 ± 6.390). Hence, both the groups have highly significant difference (t = 4.079; p = 0.0001; Fig.3).
Figure. 3. Percentage herbivoury of the Chinese grasshopper (A. cinerea) among the experimental (C. citricola’s cues treated) and control group of the Basil plant (O. basilicum) in whole plant experiment.
Note: three stars (***) show highly significant differences.
Table: 1. Summary of the percentage herbivoury of the Chinese grasshopper (A. cinerea) among the experimental (C. citricola’s cues treated) and control group of the Basil plant (O. basilicum) in three different experiments.
| % Herbivoury of the experimental (cue treated) group | % Herbivoury of the control group | p-value | |
| Single leaf laboratory experiment | 3.233 ± 1.212 | 19.47 ± 5.274 | 0.0056 |
| Single leaf plant experiment | 1.360 ± 0.536 | 22.60 ± 7.161 | 0.006 |
| Whole plant experiment | 0.4933 ± 0.179 | 26.57 ± 6.390 | 0.0001 |
Cue effectiveness
In this experiment, four different species of insects were used to check the effectiveness of the cues. For this purpose, four-round filter papers were placed in the Petri plate and the cues of the spiders were obtained by moving them on it for three days. Cue deposited filter paper and the clean filter papers were divided into two identical halves and fastened one half of the cue bearing filter paper with the other half of the cue less filter paper in the Petri plate. All the insects were put separately on different plates for thirty minutes and their time spent on both of the filter papers was calculated by stopwatch. Six readings were taken from the same specimen and plate and three specimens of each species were used for the experiment.
ANOVA test was used to compare the time spent by the Insects like Chinese grasshoppers, Acrida cinerea, bush cricket, Hexacentrus japonicus, housefly, Musca domestica, house cricket, Acheta domestica, on both clean and cue deposited filter paper. The time spent by, Acrida cinerea, on the clean filter paper was higher (1056 ± 144.5) than the cue-bearing filter paper (744 ± 144.5) (t = 1.527; p = 0.138). Similarly, the time spent by the Hexacentrus japonicus, on the clean filter paper was higher (1012 ± 160.4) than on the cue-bearing filter paper (788 ± 160.4) (t = 0.98; p-value= 0.332). The time which, Acheta domestica, on the clean area was higher (960 ± 156) than the cue bearing filter paper (840 ± 156) (t = 0.543; p = 0.590). In the case of, Musca domestica, the time spent on the clean filter paper was higher (1112 ± 128.4) than on the cue-bearing filter paper (688 ± 128.4) statistically significant difference was present among both groups (t = 2.335; p = 0.026). These differences were statistically non-significant (f = 1.116; p = 0.357; Fig. 4).
Figure 4: Comparison between the time spent on cues bearing filter paper (+) and clean filter paper (-) analyzed by ANOVA mean±SD followed by Tukey’s test between the Chinese grasshopper (G. H.), Bush-cricket (B. C.), House cricket (H. C.) and Housefly (H. F.) to evaluate the cue effectiveness (f = 1.116; p = 0.357).
- H.+ = time spent by the Chinese grasshopper on the cues bearing filter paper (744 ± 144.5).
- H.- = time spent by the Chinese grasshopper on the clean filter paper (1056 ± 144.5).
- C.+ = time spent by the bush-cricket on the cue-bearing filter paper (788 ± 160.4).
- C.- = time spent by the bush-cricket on the clean filter paper (1012 ± 160.4).
- C.+ = time spent by the house cricket on the cue-bearing filter paper (840 ± 156).
- C.- = time spent by the house cricket on the clean filter paper (960 ± 156).
- F.+ = time spent by the house fly on the cue-bearing filter paper (688 ± 128.4).
- F.- = time spent by the house cricket on the clean filter paper (1112 ± 128.4).
Note: The “ns” sign which is present on the horizontal lines shows a non-significant difference among the groups.
DISCUSSION
The grasshopper, Acrida cinerea, in our study was able to detect the cues of the spider, Cyrtophora citricola, deposited on the leaves of, Ocimum basilicum, and thus avoided them. In agroecosystems, there is a great loss to the fields due to the damage caused by the insect pests. Spiders play an important role to inhibit the harm to the plants not only by direct killing the prey but their presence also reduced the ratio of the herbivoury. Spiders produce silk and use it as a cue to deter the prey. Besides this, they also use some other cues like their waste materials, pheromones, eggs, kairomones, etc to lower the herbivoury (Abrams, 2007; Bucher et al., 2015; Cronin & Bok, 2016; Dicke & Grostal, 2001; Persons and Rypstra, 2000; Reader et al., 2016).
Predation is a great risk for the survival of insect pests in an agroecosystem. The presence of the silk of the spider acts as an obvious sign for insects to be cautious and avoid the plants to reduce the risk of predation (Tahir et al., 2019). It was also discerned in the preceding literature that the herbivores should balance the probing of good quality food and their survival rate to avoid predation. When the grasshopper was exposed to the cue’s treated (i.e., silk) leaves of the plant after a period of starvation then they ate it as no choice was given to them. In our study, a choice was given to the grasshoppers by giving them cue-treated leaves and the leaves with no cues from the spider (Cyrtophora citricola). A definite difference was noticed between the two groups after a particular time period and the grasshopper, Acrida cinerea, clearly keep away from the cues treated leaves. The insect had an approach to the visible and chemical cues of the spider. The insects were exposed to the plants after the removal of the spider so it was clear that the decline in the ratio of the herbivory was associated with the spider cues which were present thereafter their exit. This reduced tendency of the grasshopper to the cues deposited leaves was to maximize their survival by avoiding them (Bucher et al., 2015; Hlivko & Rypstra, 2003).
Spiders have gained massive importance at the industrial level due to the production of silk. The grasshoppers recognized this silk as an obvious sign of the presence of the predator and tried to avoid it. The effects of spiders other than the direct killing of the prey which is regarded as the trait-mediated effect also play a crucial role in increasing the plant biomass by lowering the herbivoury. The excretory materials of the spider, carcass of the prey and chemicals, etc are included in it. So, in the whole plant experiment when the grasshoppers were given an option among control and cue-bearing leaves within the same plant, there were a clear decline in the herbivoury was calculated (Abrams, 2007; Bucher et al., 2014b; Werner, & Peacor, 2003).
In our cue effectiveness experiment, the grasshopper was given a choice between the cue-bearing filter paper and the clean filter paper. The results showed a non-significant contrast between the time spent by the grasshopper on the clean filter paper and the cue-treated filter paper. To keep away from the threatening sites and reduce motility are included in the tactics of the prey for avoiding the predators. It was explored that due to the repetitive interaction of the grasshoppers to the cue-treated filter paper the grasshopper assessed the low predation risk by learning behaviour. The other reason for this result was that some cues of spiders were evaporative in nature so their intensity did not remain constant and reduced with the passage of time. Hence, they did not discourage the insects effectively (Bucher et al., 2014a; Kortet & Hedrick, 2004).
Another possibility of this result was the duration of the time which was given to these insects. It might be that the given duration was not adequate for them to learn avoiding behaviour. The reoccurrence of the alike incentive might also be another prospect of these results by arising the behaviour of habituation in the insects. So, when they realized about no real danger, they stopped their response to the cues. Unlike this, In the field, the grasshoppers avoided the cues as they considered them a danger to their survival so they mostly avoided them (Kortet & Hedrick, 2004; Rankin et al., 2009; Tahir et al., 2017; Tahir et al., 2019).
The results of this study clearly showed that spider cues were a good source to repel insect pests in an agroecosystem. Both the direct and indirect cues of the spider eluded the risk of foraging to an extent. There are some limitations in this study of having a short duration of time for the insects. The small number of insect specimens is also one of its obstacles. Hence, there is a need for more advanced studies to evaluate the risk of foraging and the effectiveness of the cues of different species of spiders on the different insect pests. The results of this study may be more or less affected by the surroundings as the experimental work was performed either in laboratory conditions or in a semi-field environment.
CONCLUSION
This study revealed that the cues of the spider play a vital role in increasing the plants’ biomass by acting as a threat to the insect pest. The insect pest did not forage at a large rate and hence their percentage herbivoury declined. The insect species clearly avoided their direct and indirect cues to reduce the risk of predation by exhibiting anti-predator tactics.
This study showed that different insects including grasshoppers did not avoid the spider cues if they continuously encountered the cues. It might be due to the learning behaviour of the insects as they became habituated and learned that there was no direct danger to their survival because of the absence of the spider. The time duration which was given to them to encounter the cues on the filter paper also affected their behaviour and thus they showed the non-significant results. However, these results might be affected due to the laboratory and semi-field conditions. Anyhow, spiders act as a biological agent by reducing the foraging behaviour of insect pests.