Direct Effect of Cannibalism and Intraguild Predation in Menochilus sexmaculatus (Coleoptera: Coccinellidae)¹

The cotton mealybug, Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae), is an insect pest of multiple crops in many countries worldwide (Wang et al. 2020). It can attack at least 213 host plant species within 56 families (Abdulrassoul et al. 2015). Phenacoccus solenopsis damages plants directly through feeding on any aboveground parts of the plants and indirectly by producing waxy secretions supporting sooty mold growth that hinders photosynthesis (Waqas et al. 2021). Currently, conventional insecticides are the most common and effective method used for suppressing P. solenopsis worldwide, but its insecticide resistance and tolerance have increased (Kaur and Virk 2012, Waqas et al. 2021). Therefore, biocontrol agents are being investigated as potential alternatives to control this pest (Zilaei et al. 2022).

Ladybird beetles (Coleoptera: Coccinellidae) are the dominant predator group in controlling populations of P. solenopsis in nature (Tong et al. 2019). Menochilus sexmaculatus F. (Coleoptera: Coccinellidae) is an omnivorous predator capable of feeding on many insect pests (Ranjbar et al. 2020) and has potential as a biological control candidate against P. solenopsis (Waqas et al. 2021). However, similar to other predators, the potential control capacity of M. sexmaculatus on P. solenopsis may depend not only on its fitness and predatory efficacy, but also on other factors, such as the consequence of both conspecific and heterospecific interactions with other individuals that share the same resource (Abraços Duarte et al. 2021, Burgio et al. 2002).

Conspecific and heterospecific interactions are common and important characteristics of many ladybird beetle species and then may affect the ladybird beetle population dynamics (Agarwala and Dixon 1992, Rasekh and Osawa 2020). Ladybird beetles that use the same habitat and prey are likely to compete with one another for common resources and subsequently inflict death on each other via cannibalism and intraguild predation (IGP) (Burgio et al. 2002, Lucas et al. 1998, Pervez et al. 2021, Ranjbar et al. 2020). Cannibalism can be observed in various life stages of ladybird beetles among conspecific species, including egg, larva, and pupa (Kundoo and Khan 2017, Pervez et al. 2021, Yasuda et al. 2001). Intraguild predation is a type of predation in which predators feed on competitors within a guild (Polis and Holt 1992). These two types of interactions can promote the sustenance of the predator at prey shortage scenarios or in the absence of high-quality prey conditions, or eliminate the interacting species (Momen and Abdel-Khalek 2021, Shakya et al. 2010). The impacts of cannibalism and IGP on ladybird beetle population dynamics are diverse (Agarwala and Dixon 1992, Takizawa and Snyder 2011, Yasuda et al. 2001) and may be influenced by the different vulnerabilities and aggressiveness of ladybird beetles to conspecific and heterospecific species. For example, Harmonia axyridis (Pallas) has been shown to be more intensely aggressive toward H. yedoensis Takizawa than to conspecific species, whereas H. yedoensis shows similar aggressiveness to both conspecific and heterospecific individuals. The intense characteristic in IGP and less so in cannibalism in H. axyridis may partly cause it to be the dominated predator of aphidophagous guilds, but a similar intensity in IGP and cannibalism in H. yedoensis may limit its population densities (Rasekh and Osawa 2020).

In many crops, M. sexmaculatus is known to prey on P. solenopsis together with other ladybird beetle species, including H. axyridis (Xu et al. 2017). Cannibalism and IGP were frequently observed on M. sexmaculatus (Pervez et al. 2021, Ranjbar et al. 2020, Yadav et al. 2019). However, information regarding the occurrence of IGP between H. axyridis and M. sexmaculatus, and the differences of the aggressiveness or vulnerability of M. sexmaculatus to conspecific and heterospecific species are lacking. The aims of this study were to (1) investigate the occurrence of IGP between M. sexmaculatus and H. axyridis, and (2) compare the aggressiveness or vulnerability of different life stages of M. sexmaculatus when encountering all stages of M. sexmaculatus or H. axyridis.

Menochilus sexmaculatus colonies were collected from common bean fields at the Experimental Farm, South China Agricultural University (Guangzhou, Guangdong, China) in August 2021. Harmonia axyridis was obtained from a laboratory colony at Qingdao Agricultural University (Qingdao, Shandong, China) in August 2021. Pairs of M. sexmaculatus or H. axyridis were placed in 3-cm Petri dishes (diameter: 3 cm, height: 1.5 cm) and reared on P. solenopsis and Aphis craccivora Koch for more than 10 generations. Phenacoccus solenopsis was collected from cotton, and A. craccivora was obtained from common bean at the Experimental Farm in June 2021. Phenacoccus solenopsis and A. craccivora were maintained on cotton plants (Gossypium hirsutum L., cultivar: Lumianyan no. 32) and broad bean plants (Vicia faba L., cultivar: Jinnong), respectively. Ladybird beetle eggs were harvested and individually transferred to new Petri dishes using a fine camel hair brush. Newly hatched larvae were individually placed into a new 3-cm Petri dish and provisioned with an excess of P. solenopsis and A. craccivora daily until they reached the life stages for all subsequent experiments. All colonies and experiments were maintained in an insectary at 25 ± 1°C and 65 ± 5% relative humidity (RH) under a 16L:8D photoperiod.

The presence and strength of predation between two M. sexmaculatus individuals, or between M. sexmaculatus and H. axyridis, were examined in 3-cm Petri dishes. Cannibalism trials were conducted using the eggs, four larval instars, pupae, and female adults of M. sexmaculatus. Intraguild predation experiments were also evaluated using various life stages of M. sexmaculatus and H. axyridis. Combinations involving the life stages (egg–egg, egg–pupae, pupae–pupae) that exhibited nonpredatory interactions were not included in the study. Thus, 25 different combinations were tested for the cannibalism trials, and 45 different combinations were conducted for the IGP trials. Each combination was replicated 15 times.

Eggs, larvae, and pupae of both ladybird beetle species used in our experiments were < 24 h old, and the adult females used were 1–2 wk old. Before testing, the ladybird beetles were isolated with a water-saturated cotton ball in a 3-cm Petri dish for 12 h to standardize their hunger level. Then, individuals of M. sexmaculatus were confined in a 3-cm Petri dish together with one individual from M. sexmaculatus or H. axyridis colonies. After 24 h, the Petri dish was checked under a stereo microscope to determine the survival of both ladybird beetles. To assess the natural mortality levels of each ladybird beetle species during a 24-h period, 15 individuals of each ladybird beetle species at different life stages were individually confined in the Petri dish containing a water-saturated cotton ball. In the case of interactions with eggs and pupae, larvae hatching from eggs or adult emergence from pupae that had been exposed to a predator were compared with those of the control treatment to determine the incidence of predation.

Statistical analyses were conducted using SPSS (version 21.0; SPSS, Inc., Chicago, IL). Data on the number of individuals consumed in each combination in cannibalism between two M. sexmaculatus individuals and IGP between M. sexmaculatus and H. axyridis were compared using a χ² test (two-tailed Fisher’s exact test, P < 0.05). The same χ² test was also used to compare differences between the number of M. sexmaculatus consumed by M. sexmaculatus and H. axyridis, and the number of M. sexmaculatus and H. axyridis eaten by M. sexmaculatus.

Natural mortality in the control treatments was low (from 0 to 6.7%) for all life stages of M. sexmaculatus and H. axyridis. Since both the eggs and pupae of M. sexmaculatus and H. axyridis could not exhibit predatory capacity, they were the victims of predation in all combinations. Additionally, when M. sexmaculatus adults were paired with four larval instars and adults of M. sexmaculatus or H. axyridis, no consumption was observed on the ladybird beetle adults.

In combinations with M. sexmaculatus eggs, 86.67%, 93.33%, 93.33%, 100%, and 100% of eggs were consumed by first to fourth instars and adults of M. sexmaculatus, respectively. When M. sexmaculatus pupae were provided, only 6.67%, 13.33%, and 20% of M. sexmaculatus pupae were consumed by the third and fourth instars and adults of M. sexmaculatus, respectively. Except for the combination when the fourth instars of M. sexmaculatus were paired with M. sexmaculatus adults, later stages of M. sexmaculatus larvae and adults fed on earlier stages of M. sexmaculatus larvae than the reverse (Fisher’s exact test: P < 0.05 for all). In combination with fourth instars of M. sexmaculatus, the number of fourth instars of M. sexmaculatus consumed by M. sexmaculatus adults was not significantly different than the reverse (Fisher’s exact test: P = 0.100) (Fig. 1). When two same instar larvae were placed together, cannibalism rates were 40%, 53.33%, 33.33%, and 60% from first to fourth instars, respectively.

When eggs of M. sexmaculatus or H. axyridis were provided, both larvae and adults of H. axyridis (first instars: 60%; second instars: 60%; third instars: 100%; fourth instars: 93.33%; adults: 93.33%) and M. sexmaculatus (larvae: 100%; adults: 100%) acted as intraguild (IG) predators. Only third and fourth instars and adults of H. axyridis could feed on M. sexmaculatus pupae (third instars: 66.67%; fourth instars: 86.67%; adults: 60%), but H. axyridis pupae were not killed by M. sexmaculatus. In combinations with first and second instars of M. sexmaculatus, M. sexmaculatus larvae were more vulnerable to IGP by H. axyridis than vice versa in all cases (Fisher’s exact test: P < 0.05 for all), except for when the second instars of M. sexmaculatus were paired with the first instar H. axyridis larvae (Fisher’s exact test: P = 0.651). Third instars of M. sexmaculatus fed on more first instars of H. axyridis than the reverse (Fisher’s exact test: P < 0.05), whereas they were more vulnerable to IGP by third and fourth instars and adults of H. axyridis than vice versa (Fisher’s exact test: P < 0.05 for all). The number of third instars of M. sexmaculatus and second instars of H. axyridis that were consumed by each other did not differ significantly (Fisher’s exact test: P = 0.245). Fourth instars of M. sexmaculatus preyed on more first and second instars of H. axyridis than vice versa (Fisher’s exact test: P < 0.05 for both). Fourth instars of M. sexmaculatus were more vulnerable to IGP by fourth instars and adults of H. axyridis than the reverse (Fisher’s exact test: P < 0.05 for both), whereas the number of third instars of H. axyridis and fourth instars of M. sexmaculatus consumed by each other was not significantly different (Fisher’s exact test: P = 0.700). Menochilus sexmaculatus adults fed on more first and second instars of H. axyridis than vice versa (Fisher’s exact test: P < 0.05 for both). However, the number of third or fourth instars of H. axyridis and adults of M. sexmaculatus consumed by each other was not significantly different (Fisher’s exact test: P > 0.05 for both) (Fig. 2).

First instars of M. sexmaculatus could not prey on second, third, and fourth instars, and pupae of M. sexmaculatus and H. axyridis. First instars of M. sexmaculatus consumed significantly more first instars of M. sexmaculatus than first instar H. axyridis larvae (Fisher’s exact test: P = 0.017), but no significant differences were observed between eggs of M. sexmaculatus and H. axyridis (Fisher’s exact test: P = 0.215). In combination with second instars of M. sexmaculatus, none of the third and fourth instars, and pupae were the victims of predation. Second instars of M. sexmaculatus consumed significantly more first instars of M. sexmaculatus than first-instar H. axyridis larvae (Fisher’s exact test: P = 0.003); no significant differences were observed between eggs or second instars of M. sexmaculatus and H. axyridis (Fisher’s exact test: P > 0.05 for both). When paired with a conspecific third instar larva or a heterospecific third instar larva together, third instars of M. sexmaculatus killed more conspecific larvae (Fisher’s exact test: P = 0.042). However, there were no differences in the feeding events between eggs, first instars, second instars, or pupae of M. sexmaculatus and H. axyridis (Fisher’s exact test: P > 0.05 for all). Additionally, third instars of M. sexmaculatus could not consume the fourth instars of M. sexmaculatus and H. axyridis. Fourth instars of M. sexmaculatus consumed significantly more fourth instars of M. sexmaculatus than fourth instar H. axyridis larvae (Fisher’s exact test: P = 0.021), but no significant differences were found between eggs, first instars, second instars, third instars, or pupae of M. sexmaculatus and H. axyridis (Fisher’s exact test: P > 0.05 for all). When paired with M. sexmaculatus adults, a higher number of predation events on third instars of M. sexmaculatus was observed than on third instar H. axyridis larvae (Fisher’s exact test: P < 0.001). However, no significant differences were observed between eggs, first instars, second instars, fourth instars, or pupae of M. sexmaculatus and H. axyridis (Fisher’s exact test: P > 0.05 for all) (Fig. 3).

For eggs and first instars of M. sexmaculatus, no statistical differences were found in the number of M. sexmaculatus consumed between individuals of the same developmental stage of M. sexmaculatus and H. axyridis (Fisher’s exact test: P > 0.05 for all). With second instars of M. sexmaculatus, the number of M. sexmaculatus larvae consumed by third instars and adults of H. axyridis was significantly higher than those by third instars and adults of M. sexmaculatus (Fisher’s exact test: P < 0.05 for both). However, there were no significant differences in the feeding events on M. sexmaculatus between the first, second, or fourth instars of M. sexmaculatus and H. axyridis (Fisher’s exact test: P > 0.05 for all). Third instars of M. sexmaculatus could not be consumed by the first instars of M. sexmaculatus and H. axyridis. The number of third-instar M. sexmaculatus larvae consumed by third and fourth instars of H. axyridis was significantly higher than those by third and fourth instars of M. sexmaculatus (Fisher’s exact test: P < 0.05 for both). Nevertheless, no significant differences were found in feeding events on M. sexmaculatus between second instars or adults of M. sexmaculatus and H. axyridis (Fisher’s exact test: P > 0.05 for both). Neither of the first and second instars of M. sexmaculatus and H. axyridis could feed on the fourth instars of M. sexmaculatus. The number of M. sexmaculatus larvae consumed by third instars and adults of H. axyridis was significantly higher than those by third instars and adults of M. sexmaculatus (Fisher’s exact test: P < 0.05 for both). However, when paired with a conspecific fourth-instar larva or a heterospecific fourth-instar larva together, there were no significant differences in the feeding events on fourth instars of M. sexmaculatus between these two species (Fisher’s exact test: P = 0.215). Neither of the first and second instars of M. sexmaculatus and H. axyridis could consume M. sexmaculatus pupae. The number of M. sexmaculatus pupae consumed by third and fourth instars of H. axyridis was significantly higher than those by third and fourth instars of M. sexmaculatus (Fisher’s exact test: P < 0.05 for both). However, no statistical differences were found in the feeding events on M. sexmaculatus pupae between adults of M. sexmaculatus and H. axyridis (Fisher’s exact test: P = 0.060) (Fig. 4).

Our results demonstrated that M. sexmaculatus could feed on conspecific individuals and was the IG predator and/or intraguild prey (IG prey) in confrontations with H. axyridis. Success of cannibalism and IGP was stage dependent: young stages of ladybird beetles were the most susceptible to predation, and relatively older larvae and adults of ladybird beetles were likely to be predators in most cases. In the IGP assays with the same instar larvae, H. axyridis were always victorious compared to M. sexmaculatus. Additionally, M. sexmaculatus intensively killed the conspecific individuals over heterospecific competitors, whereas they were more vulnerable to heterospecific individuals than to conspecific ladybird beetles.

In the present study, M. sexmaculatus could act as predator and/or prey when confronted with conspecific and heterospecific ladybird beetle individuals, which is consistent with previous studies (Pervez et al. 2021, Ranjbar et al. 2020). In addition, the early life stages of M. sexmaculatus and H. axyridis, which are the smaller-sized individuals, were vulnerable to cannibalism and IGP. Generally, cannibalism and IGP between predators are affected by the body size of species, in which the smaller individuals are consumed by larger ones (Abraços Duarte et al. 2021, Lucas et al. 1998, Rasekh and Osawa 2020). However, larger individuals did not possess an absolute advantage in all combinations investigated in the study. When the size of M. sexmaculatus larvae were larger than those of the H. axyridis with which they were paired (M. sexmaculatus × H. axyridis: second instars × first instars, third instars × second instars, fourth instars × third instars), they acted as both the IG predator and IG prey. Moreover, in confrontations with the same instar larvae, H. axyridis was always victorious. The superiority of H. axyridis could be attributed to its greater aggressiveness toward heterospecific species (Katsanis et al. 2013, Rasekh and Osawa 2020, Yasuda et al. 2001). In addition, because the defensive chemical compounds in the hemolymph and reflex fluid of H. axyridis larvae are repulsive or toxic to many ladybird beetle species, this may be the way in which H. axyridis avoids IGP by other ladybird beetle species (Cottrell 2004, Pell et al. 2008). In our experiment, the species-specific chemical protection may help H. axyridis against M. sexmaculatus. Nevertheless, adults of H. axyridis and M. sexmaculatus were not preyed upon by the other ladybird beetle individuals. Body sclerotization of ladybird beetle adults may be a reason for low predation on this prey type, as predators of aphids have an obvious preference for soft-bodied insects (De Clercq et al. 2003). Moreover, because eggs and pupae cannot exhibit escape behavior, they are only the victims of predation. Similar results were observed in other studies reporting cannibalism and IGP could be affected by the mobility of predators (Lucas et al. 1998, Pervez et al. 2021).

Generally, the incidence of cannibalism or IGP of ladybird beetles increases when the relative abundance of shared prey to ladybird beetle is low or in the absence of high-quality prey scenarios (Agarwala and Dixon 1992, Kundoo and Khan 2017, Ranjbar et al. 2020, Sato et al. 2003). The conspecific and heterospecific predators can act as an alternative prey source to the organism under these unfavorable conditions (Agarwala and Dixon 1992). However, this is not always beneficial when the prey are full siblings (Osawa 1992), or the heterospecific ladybird beetles are toxic (Agarwala and Dixon 1992, Sato and Dixon 2004). Many studies have shown that H. axyridis is unsuitable prey for multiple ladybird beetle species (Cottrell 2004, Katsanis et al. 2013, Rasekh and Osawa 2020). In the present experiments, the intensity of predation on M. sexmaculatus was higher than that on H. axyridis when M. sexmaculatus was the predator. In the scarce prey conditions or without high-quality prey scenarios, M. sexmaculatus may have to concentrate on consuming conspecific ladybirds to ensure its survival and be reluctant to consume H. axyridis. This intense characteristic in cannibalism and less so in IGP in M. sexmaculatus may negatively affect its population densities. In addition, by comparing the predation incidence between IGP by H. axyridis and cannibalism by M. sexmaculatus, we found that M. sexmaculatus was highly susceptible to predation by H. axyridis. The greater aggressiveness of H. axyridis toward M. sexmaculatus may further reduce the population densities of M. sexmaculatus when food resource conditions are unfavorable. Consequently, H. axyridis may restrict the population densities of M. sexmaculatus by its high aggressiveness and low vulnerability, which may influence the population establishment and subsequent application of M. sexmaculatus in biological control programs.

However, we must note that this study was conducted in an oversimplified, confined area. In the field, many factors may decrease the encounter rate between different predators and reduce the intensity of cannibalism and IGP, such as the density of prey resources (Abraços Duarte et al. 2021, Burgio et al. 2002, Lucas et al. 1998), habitat structure (Janssen et al. 2007, Sun et al. 2021), and emigration time (Sato et al. 2003). Thus, our laboratory studies may overestimate the effects of cannibalism and IGP on M. sexmaculatus under natural conditions. Despite this, simplified laboratory-based trials remain essential for depicting the interspecific relationship between M. sexmaculatus and H. axyridis and investigating the potential impacts of cannibalism and IGP on M. sexmaculatus.

In conclusion, M. sexmaculatus could prey on conspecific individuals and acted as an IG predator and/or IG prey when confronted with H. axyridis. Moreover, M. sexmaculatus was more intensely aggressive toward conspecific individuals and less so to heterospecific ladybirds, but was more susceptible to H. axyridis than to M. sexmaculatus. It is likely that the establishment of M. sexmaculatus populations will be negatively affected due to the presence of H. axyridis, which may hinder the success of biological control programs in the management of P. solenopsis populations in crops. Given the limitations of our laboratory setting, further research should be conducted under more realistic conditions in the field and semi-field levels to understand how species interactions may influence the short- and long-term population dynamics of M. sexmaculatus and what measures may effectively contribute to conserving the populations of M. sexmaculatus.

The authors thank Yi Zhang (Qingdao Agricultural University) for providing the ladybird beetles. This study was supported by the Laboratory of Lingnan Modern Agriculture Project (NT2021003), the National High-Level Talent Special Support Plan (2020), and the National Key Research and Development Program of China (2017YFD0200400).