Introduction
The family Chrysopidae includes approximately 1200 species. The larvae of all species are voracious predators and feed on key pests of crops such as aphids, mealybugs, lepidopteran and coleopteran eggs, thrips and spider mites (Canard et al., Reference Canard, Séméria and New1984; McEwen et al., Reference McEwen, New and Whittington2001). In addition, chrysopids of certain genera (e.g. Chrysopa) are voracious during the adult stage as well (Brooks and Barnard, Reference Brooks and Barnard1990). Chrysopids are widespread in several habitats including agroecosystems and forests (McEwen et al., Reference McEwen, New and Whittington2001). Particularly, because of the occurrence of several species in agricultural systems and due to the voracity of certain species, green lacewings of the family Chrysopidae are considered important biological control agents of key pests (Tauber et al., Reference Tauber, Tauber, Daane and Hagen2000; Pappas et al., Reference Pappas, Broufas and Koveos2011). Despite the fact that many chrysopids are well known for their high predation efficiency against several pests, only a few species are currently commercially available, with Chrysoperla carnea sensu lato being the most widely used in biological control (Pappas et al., Reference Pappas, Broufas and Koveos2011). Concretely, among the 20 described species of the genus Chrysoperla, ‘Chrysoperla carnea’ is the only species marketed by the European biocontrol industry for aphid control. Yet, a number of other Chrysoperla species may prove more efficient/suitable than the commercial C. carnea in controlling different pests under specific conditions.
Our previous work on the European species of the C. carnea complex has revealed their potential as biological control agents for use in central and southern Europe (Pappas et al., Reference Pappas, Karagiorgou, Papaioannou, Koveos and Broufas2013; Athanasiadis et al., Reference Athanasiadis, Pappas and Broufas2021). These lacewings are reproductively isolated species that can be distinguished by the low-frequency species-unique courtship songs they produce by vibrating their abdomen on the substrate before mating (Brooks, Reference Brooks1994; Henry et al., Reference Henry, Brooks, Thierry, Duelli, Johnson, McEwen, New and Whittington2001; Noh and Henry, Reference Noh and Henry2010; Henry et al., Reference Henry, Brooks, Duelli, Johnson, Wells and Mochizuki2013). Among the five European species of the C. carnea group, Chrysoperla agilis has been recorded in southern Europe, southwestern Asia to northern Iran and central Alaska (Henry et al., Reference Henry, Brooks, Duelli and Johnson2003; Henry et al., Reference Henry, Brooks, Johnson, Wells and Duelli2011). C. agilis is able to survive and reproduce in a wide range of temperature regimes and larvae prey upon factitious foods such as Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) eggs in the lab (Pappas et al., Reference Pappas, Karagiorgou, Papaioannou, Koveos and Broufas2013; Athanasiadis et al., Reference Athanasiadis, Pappas and Broufas2021). These findings, along with its wide distribution and habitat range and occurrence in agricultural crops (Henry et al., Reference Henry, Brooks, Duelli and Johnson2003; Henry et al., Reference Henry, Brooks, Johnson, Wells and Duelli2011), render C. agilis a promising candidate for mass-rearing and use in augmentative biological control.
Chrysoperla mutata (McLachlan) is not a member of the carnea complex but of the pudica group (Brooks, Reference Brooks1994; Duelli, Reference Duelli, McEwen, New and Whittington2001). With a distribution ranging from Africa to the Mediterranean Europe and Asia, and lately in Russia and Tenerife (Thierry et al., Reference Thierry, Canard and Mirmoayedi2004; Canard and Thierry, Reference Canard and Thierry2013; Duelli et al., Reference Duelli, Monserrat and Henry2019; Makarkin and Shchurov, Reference Makarkin and Shchurov2019) it is considered to be able to survive and reproduce in areas with dry and hot summers as well as mild winters (Szentkirályi, Reference Szentkirályi, McEwen, New and Whittington2001). As with the carnea cryptic species, the biology and ecology of C. mutata as well as its predation efficiency against crop pests remain largely unexplored (Canard and Thierry, Reference Canard and Thierry2013).
The two lacewings show overlapping distribution in southern Europe, and in addition, C. agilis can be found in northern habitats as well. Moreover, our experience suggests that the rearing of C. mutata in large numbers is feasible due to its low occurrence of cannibalism as well as the wide prey/food range. Given the need to increase biocontrol solutions against key pests of crops and the potential of both C. agilis and C. mutata for use in augmentative biocontrol, we here evaluated the predation efficiency and ability of both species to suppress the populations of the green peach aphid Myzus persicae and the cotton mealybug Phenacoccus solenopsis on sweet pepper plants. Both pest species cause serious damage to many crops including greenhouse sweet pepper (van der Ent et al., Reference van der Ent, Knapp, Klapwijk, Moerman, van Schelt, de Weert, Girard and Stroobach2017; Pekas et al., Reference Pekas, De Craecker, Boonen, Wäckers and Moerkens2020; Bragard et al., Reference Bragard, Di Serio, Gonthier, Jaques Miret, Justesen, Magnusson, Milonas, Navas-Cortes, Parnell, Potting, Reignault, Thulke, Van der Werf, Civera, Yuen, Zappalà, Gregoire, Malumphy, Campese, Czwienczek, Kertesz, Maiorano and MacLeod2021). The results will be useful to assess the use of C. agilis and C. mutata in applied biocontrol programmes.
Materials and methods
Laboratory rearing of green lacewings
The laboratory colonies of C. agilis and C. mutata were maintained at 25 + 1°C and 16:8 LD. The colony of C. mutata was established with adults collected in the area of Chalkidiki (Northern Greece), whereas C. agilis was collected in Crete (Southern Greece). The larvae of both species were fed on frozen E. kuehniella eggs and adults were maintained in cylindrical plastic cages (35 cm in height × Ø25 cm), as described by Pappas et al. (Reference Pappas, Broufas and Koveos2007). Briefly, adults were reared in groups of females and males in cages and fed with a protein-based liquid food (a volumetric mixture of honey, sugar, yeast hydrolysate and water, 1:1:1:1) applied daily on the top of the mesh covering each cage. Lacewing identification was performed by song analysis and morphological traits by Prof. Charles S. Henry (University of Connecticut).
Experimental green lacewing larvae
For the experiments, different instars of lacewing larvae were used. For this purpose, eggs were collected at 24 h intervals from the lacewing colonies and maintained individually in Petri dishes until hatching. Afterwards, the larvae were reared until the desired instar (second or third, depending on the experiment, see below) with ad libitum access to E. kuehniella eggs.
Herbivores used as prey in the experiments
Two different prey species were tested in the experiments: (a) the aphid M. persicae that originated from tobacco in the area of Komotini (Northern Greece) and the mealybug P. solenopsis that was collected from sweet pepper plants in the area of Crete. Both species were reared on potted sweet pepper (Capsicum annuum L.) plants under laboratory conditions (25 ± 2°C, 65 ± 5% RH, 16:8 LD).
Plants used in the experiments
Pepper (C. annuum L., cv P13) seedlings were transplanted in plastic pots (0.5 litres) filled with peat (Klasmann-Deilmann GmbH). Plants were maintained in a growth chamber (25 ± 2°C, 65 ± 5% RH and 16:8 LD) and were watered and fertilized once per week (N-P-K, 10-10-10, 1 g l−1). For the experiments, we used plants 4-weeks after transplantation (d28).
Predation efficiency against aphids and mealybugs
Prey consumption by C. agilis and C. mutata when provided aphids or mealybugs was assessed in the laboratory and the greenhouse.
Laboratory trials
In the laboratory, daily prey consumption by third-instar larvae of C. agilis and C. mutata was assessed in Petri dish assays at 25 ± 1°C, 16:8 LD. The larvae were placed individually in Petri dishes (Ø5 cm) without food for 24 h. Afterwards, each lacewing larva was offered 200 (third–fourth instar) aphid (M. persicae) nymphs or 150 newly hatched mealybug (P. solenopsis) nymphs. Prey consumption was recorded after 24 h. Each treatment included 15 replicates (lacewing larvae).
Greenhouse experiments
In the greenhouse, 60 potted pepper plants were infested with either 100 aphid (third–fourth instar) nymphs or one mealybug female carrying an egg-sac (d28). The plants were placed individually in insect-proof cages (60 × 33 × 33 cm3) (25 ± 1°C, 16:8 LD). Forty plants were assigned to the predator treatment (i.e. 20 repetitions per predator). For this, ten lacewing larvae from each lacewing species (second instar) were transferred onto each plant, both 3 days after the aphid infestation (d31) and again 1 week later (i.e. 20 lacewing larvae were released per plant in total). Lacewing larvae had been fed with E. kuehniella eggs until they reached the second larval instar. Another 20 plants were infested with aphids without predators being released (control treatment). Aphid numbers per plant were assessed 1 week after the second release (d42) both in the predator and control plants (fig. 1a).
Figure 1. Experimental set-up used to evaluate the predation efficiency of larvae of C. mutata and C. agilis on pepper plants in insect cages infested by (i) M. persicae or (ii) P. solenopsis. Pest suppression was assessed 11 or 21 days after lacewing (second-instar larvae) initial release on the plants for aphids and mealybugs, respectively.
For the mealybug experiments, 60 plants were infested with one mealybug female carrying an egg-sac (d0). Forty plants were assigned to the predator treatment, where 15 lacewing larvae (second instar) of each lacewing species were transferred on each plant after 1 (d35), 2 (d42) and 3 weeks (d49) (i.e. 45 lacewing larvae per plant in total). Lacewing larvae had been fed with E. kuehniella eggs until the second larval instar. Another 20 plants were infested with mealybug without predators being released (control treatment). Mealybug numbers per plant were assessed 1 week after the last predator release (d56), both in the predator and control plants (fig. 1b).
Lacewing individuals (i.e. emerging adults and live lacewing pupae) inside each cage were also recorded per plant (insect cage).
Data analysis
To compare daily prey consumption between the different lacewing species a t-test was used. In the greenhouse experiment, failing to meet the requirements for parametric analysis, means were compared with the non-parametric Kruskal–Wallis test, followed by pairwise comparisons with Dunn's test. Statistics were performed using SPSS (2020).
Results
Short-term prey consumption
Prey consumption by third-instar lacewing larva in 24 h was 104.3 ± 4.4 and 103.5 ± 3.6 mealybug individuals for C. agilis and C. mutata, respectively, and 116.0 ± 4.9 and 118.2 ± 4.9 aphid individuals for C. agilis and C. mutata, respectively (fig. 2). No significant differences were recorded in the mean numbers of mealybugs (t = 0.153; df = 28; P = 0.88; fig. 2a) or aphids (F = 0.316; df = 28; P = 0.754; fig. 2b) consumed by C. agilis and C. mutata.
Figure 2. Mean number of (a) mealybugs and (b) aphids consumed by C. mutata (CM) and C. agilis (CA) third-instar lacewing larvae in 24 h in laboratory Petri dish assays (25 ± 1°C, 16:8 LD). ns: P > 0.05.
Pest suppression in the greenhouse
Aphid population levels were significantly affected by the release of the lacewing larvae (χ2 = 49.64; df = 2; P < 0.001). Mean number of aphids per plant was 4146.2 ± 151.3 individuals in control plants and decreased to 48.9 ± 5.5 and 11.4 ± 2.3 aphids after releasing C. mutata and C. agilis larvae, respectively (fig. 3a). Out of the initial number of released lacewings (20 larvae in total per plant), a low number of lacewing individuals per species (3.0 ± 1.3 and 2.6 ± 1.2 pupae per cage for C. mutata and C. agilis, respectively) was recorded in each cage for both species (U = 162.0; Z = −1.067; P = 0.286; fig. 3b).
Figure 3. Mean number of (a) aphids 14 days (d42) after plant infestation with 100 aphid (third–fourth instar) nymphs followed by the release of C. mutata (CM) or C. agilis (CA) second-instar larvae (ten larvae were released per species twice on each plant at d31 and d35, i.e. 20 larvae in total) as compared to control (CON) plants (no lacewing release), and (b) live individuals (i.e. emerging lacewing adults and live pupae) per lacewing species in each cage, Kruskal–Wallis multiple comparison analysis with a Dunn's post-hoc test. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001.
The level of mealybug infestation was significantly affected by the release of the lacewing larvae as well (χ2 = 39.403; df = 2; P < 0.001). The mean number of mealybugs per plant was 808.4 ± 55.3 individuals in control plants, which decreased to 208.5 ± 26.7 and 201.2 ± 25.5 mealybugs after releasing C. mutata and C. agilis larvae, respectively (fig. 4a). Out of the initial number of released lacewings (45 larvae in total per plant), the number of emerging lacewing adults and live pupae was 7.8 ± 0.7 and 3.2 ± 0.3 for C. mutata and C. agilis respectively (U = 29.5; Z = −4.655; P < 0.001; fig. 4b).
Figure 4. Mean number of (a) mealybugs 28 days (d56) after plant infestation with one mealybug female carrying an egg-sac followed by the release of C. mutata (CM) or C. agilis (CA) second-instar larvae (per species, 15 larvae released thrice on each plant at d35, d42 and d49, i.e. 45 larvae in total) as compared to control (Con) plants (no lacewing release), and (b) live individuals (i.e. emerging lacewing adults and live pupae) per lacewing species in each cage, Kruskal–Wallis multiple comparison analysis with a Dunn's post-hoc test. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001.
Discussion
In the present study, we assessed the ability of two green lacewing species of the genus Chrysoperla to consume individuals of two key pests of vegetable crops, the green peach aphid M. persicae and the mealybug P. solenopsis. Furthermore, we compared the two lacewings for their efficacy in suppressing the populations of these two pests when infesting pepper plants in the greenhouse. We found that both lacewings can similarly consume individuals of the green peach aphid as well as mealybugs. In addition, both were shown to be efficient in suppressing aphid and mealybug populations in the greenhouse, relative to a predator-free control.
Short-term consumption of aphids and mealybugs was similar between the two lacewing species. Both C. agilis and C. mutata third-instar larvae were shown to be able to consume a relatively high number of aphids (approximately 120 nymphs per larva) within 24 h. These results confirm the high voracity of both lacewing species and confirm what is already known about the predation efficiency of late instar larvae of Chrysoperla species (Canard and Principi, Reference Canard, Principi, Canard, Semeria and New1984; Pappas et al., Reference Pappas, Broufas and Koveos2011). Notably, both lacewings were also capable of consuming a high number of mealybug individuals, which can be an additional asset of these chrysopid species, which are largely seen as aphidophagous predators. Other Chrysoperla species have previously been recorded as natural enemies of different mealybug species in agroecosystems (McEwen et al., Reference McEwen, New and Whittington2001). However, the suitability of mealybugs to support the development and survival of lacewings should be further explored. For example, the citrus mealybug Planococcus citri was found to be suboptimal as prey for Chrysoperla lucasina, another European species of the carnea group (Messelink et al., Reference Messelink, Vijverberg, Leman and Janssen2016). Similarly, different aphid species may differentially affect the life-history traits of chrysopids, as was shown for example in the case of Pseudomallada prasinus and Chrysoperla sinica (Pappas et al., Reference Pappas, Broufas and Koveos2007; Khuhro et al., Reference Khuhro, Chen, Zhang, Zhang and Wang2012).
On the other hand, the (long-term) pest suppression in the greenhouse was found to be remarkably efficient for both lacewing species. Aphid populations were suppressed, with C. agilis being slightly more efficient than C. mutata with approximately 10 vs. 50 aphids found alive per plant, respectively as compared to the control (approximately 4000 aphids per plant). With regard to the suppression of mealybug populations in the greenhouse, both C. agilis and C. mutata larvae resulted in a reduced mealybug population growth, relative to the predator-free control, with no significant differences between the two lacewings. Hence, our results highlight the potential of both lacewing species as aphid and mealybug predators. At this point, it is important to consider that pepper plants used in our experiments were infested with a quite high number of pest individuals (100 third–fourth-instar aphid nymphs or one mealybug female carrying an egg-sac) suggesting that an earlier release at lower pest infestations could have resulted in a more pronounced reduction or even the complete pest elimination. Furthermore, it is also important to acknowledge that the releases of the predators took place in cages that may have facilitated the encounters and eventually consumption of the prey. More experiments under more realistic field conditions are needed to better evaluate the potential of C. agilis and C. mutata to control aphids and mealybugs in sweet pepper.
The reduction in aphid populations achieved by C. agilis larvae was more pronounced when compared to C. mutata. This can be explained by species-specific differences in the predation potential of each instar or the developmental rates of the two lacewings. While no studies have assessed the life-history traits of C. mutata so far, second-instar larvae of C. agilis require approximately 11 days to complete development when fed with E. kuehniella eggs at 27°C (Pappas et al., Reference Pappas, Karagiorgou, Papaioannou, Koveos and Broufas2013) suggesting that most of the released larvae (20 larvae per plant) may have pupated by the recording day. On the other hand, in the case of the mealybugs experiment that ended 2 weeks later, possibly most of the larvae released in the first two time points (in total 30 larvae per plant in d35 and d42) of both C. agilis and C. mutata may have pupated/reached adulthood. Nevertheless, a significantly higher number of C. mutata as compared to C. agilis individuals was recorded on mealybug-infested plants (approx. eight vs. three individuals) 3 weeks after the initial release of second-instar larvae, whereas this difference was not seen 11 days after the release of second-instar larvae on aphid-infested plants. Further experiments are required to verify these differences and assess the population growth of the two lacewings on plants infested by each pest.
Despite the wide distribution of C. agilis and C. mutata, relatively little is known about their feeding habits, life-history traits and ability to suppress pest populations. Both species show a preference for Mediterranean climates (Szentkirályi, Reference Szentkirályi, McEwen, New and Whittington2001; Henry et al., Reference Henry, Brooks, Duelli and Johnson2003) and under these conditions are capable of consuming aphid and mealybug individuals and suppressing their populations. Hence, both species can be promising biological control agents in warmer and dry parts of their distribution range, where other species of the -carnea group are not common (Tauber et al., Reference Tauber, Tauber, Daane and Hagen2000; Henry et al., Reference Henry, Brooks, Duelli and Johnson2003; Canard and Thierry, Reference Canard and Thierry2013; Pappas et al., Reference Pappas, Karagiorgou, Papaioannou, Koveos and Broufas2013; Duelli et al., Reference Duelli, Monserrat and Henry2019; Athanasiadis et al., Reference Athanasiadis, Pappas and Broufas2021). With regard to their feeding habits, C. agilis was previously shown to prey on E. kuehniella eggs (Pappas et al., Reference Pappas, Karagiorgou, Papaioannou, Koveos and Broufas2013), whereas there is only limited information on C. mutata larval ability to consume aphids such as Lipaphis erysimi and the spider mite Tetranychus turkestani (Zhang, Reference Zhang2003; Abdulhay, Reference Abdulhay2021). Furthermore, we show in the present study that C. mutata can be reared on E. kuehniella eggs, while preying on M. persicae and P. solenopsis nymphs.
Previous studies suggest that C. agilis and C. mutata could be mass-reared for biocontrol purposes by adopting the methods and techniques currently used for C. carnea s.l. because of the ability of both lacewings to develop and reproduce on factitious foods such as E. kuehniella eggs and their adults' non-predatory diet of sugar-rich liquids (Pappas et al., Reference Pappas, Broufas and Koveos2011; Athanasiadis et al., Reference Athanasiadis, Pappas and Broufas2021). Currently, C. carnea s.l. is the main chrysopid species used in biological control, whereas difficulties in the identification of the different species of the carnea group have been related to varying biocontrol success in the field (Tauber et al., Reference Tauber, Tauber, Daane and Hagen2000; Pappas et al., Reference Pappas, Broufas and Koveos2011; Henry et al., Reference Henry, Brooks, Duelli, Johnson, Wells and Mochizuki2013). Here, we revealed the biocontrol potential of C. agilis which is distinguishable from the other cryptic species by the distinct mating signals of adults (Henry et al., Reference Henry, Brooks, Duelli and Johnson2003; Noh and Henry, Reference Noh and Henry2010). Also, C. mutata produces different mating signals and bears distinct morphological traits from C. pudica (Duelli et al., Reference Duelli, Monserrat and Henry2019). The predation efficiency of C. carnea s.l. has been studied against different aphid and mealybug species (e.g. Hagley, Reference Hagley1989; Atlihan et al., Reference Atlihan, Kaydan and Özgökçe2004; El-Sahn and Gaber, Reference El-Sahn and Gaber2012; Shrestha and Enkegaard, Reference Shrestha and Enkegaard2013; Jessie et al., Reference Jessie, Giles, Rebek, Payton, Jessie and McCornack2015; Saljoqi et al., Reference Saljoqi, Asad, Khan, Haq, Nasir, Zada, Ahmad, Nadeem, Huma and Salim2016). To date, no study had assessed its biocontrol efficiency against M. persicae or P. solenopsis in pepper by C. agilis and C. mutata. Considering the preference of the latter for warmer and drier habitats (Canard and Thierry, Reference Canard and Thierry2013), comparative studies with the commercial C. carnea s.l. could reveal advantages of C. agilis and C. mutata in aphid and mealybug biocontrol under warm and dry conditions.
In conclusion, both C. agilis and C. mutata are predators with high potential in biological pest control. The larvae of both were shown capable of suppressing M. persicae as well as P. solenopsis populations on pepper plants for the time period of our experiments, with C. agilis being more efficient against aphids than C. mutata. Further research focusing on the life-history traits of both chrysopids under different conditions coupled with long-term greenhouse experiments with varying densities of prey and released larvae is needed to clarify the role of C. agilis and C. mutata under realistic conditions.
Acknowledgements
We thank the undergraduate students Evangelos Doulopoulos and Konstantinos Tsimourdagkas for technical help during the course of the experiments. Part of this work was funded by Biobest Group NV (project number 82083).
Author contributions
M. P., G. B., A. P. and F. W. conceived the study. G. K. and A. S. performed the experiments. M. P., G. B. and G. K. wrote the manuscript with input from A. P. and F. W. All authors read and approved the manuscript.
Conflict of interest
A. P. and F. W. are employed at Biobest Group NV. Authors declare no conflict of interest.
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