INTRODUCTION
⌅The tomato leaf miner (TLM), Tuta absoluta (Meyrick), is an important native pest on tomato (Solanum lycopersicum L.) in South America (11. Guedes RNC, Picanço MC. The tomato borer Tuta absoluta in South America: pest status, management and insecticide resistance. Bulletin OEPP/EPPO. 2012;42:211-216. https://doi.org/10.1111/epp.2557 ). This specie was reported in eastern Spain in late 2006 (22. Urbaneja A, Vercher R, Navarro V, García MF, Porcuna JL. La polilla del tomate, Tuta absoluta. Phytoma España. 2007;194:16-23. ) and, since then, it has spread to various countries in Europe, Africa, Middle, East and Asia (33. Toševski I, Jović J, Mitrović M, Cvrković T, Krstić O, Krnjajić S. Tuta absoluta (Meyrick, 1917) (Lepidoptera, Gelechiidae): a New Pest of Tomato in Serbia. Pesticides & Phytomedicine. 2011;3:197-204. https://doi.org/10.2298/PIF1103197T , 44. Biondi A, Guedes NRC, Wan F, Desneux N. Ecology, worldwide spread, and management of the invasive south american tomato pinworm, Tuta absoluta: Past, present, and future. Annual Review of Entomology. 2018;63:239-58. https://doi.org/10.1146/annurevento-031616-034933 ). In the western hemisphere, it was reported in Panama (2011) and Costa Rica (2014) (55. CABI Head Office, Wallingford, UK. Distribution Maps of Plant Pests, Tuta absoluta. [Distribution map]. Map 723(1st revision). 2016. Consulted: 29/3/2018 available online: http://www.cabi.org/isc/datasheet/49260 ) and, more recently, in a Caribbean country such as Haiti (66. Verheggen F, Bertin R. First record of Tuta absoluta in Haiti. Entomologia Generalis. 2019; 38:349-353. https://doi.org/10.1127/entomologia/2019/0778 ). The species has a huge economic impact, and yield losses of up to 100 % and reduction in fruit quality have been reported both in open field and greenhouse crops, mainly where control methods have not been applied (77. Bueno VHP, van Lenteren JC, Lins Jr, Calixto AM, Montes F, Silva D, et al. New records of Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) predation by Brazilian Hemipteran predatory bugs. Journal Applied Entomology. 2013;137:29-34. https://doi.org/10.1111/jen.12017 , 88. Han P, Bayram Y, Shaltiel‐Harpaz L, Sohrabi F, Saji A, Esenali UT, et al. Tuta absoluta continues to disperse in Asia: damage, ongoing management and future challenges. Journal of Pest Science. 2018. https://doi.org/10.1007/s10340-018-1062-1 ).
Several approaches were implemented to manage T. absoluta in its area of origin, mainly by using insecticides, but with limited success due to insect resistance (99. Haddi K, Berger M, Bielza P, Rapisarda C, Williamson MS, Moores G, et al. Mutation in the ace-1 gene of the tomato leaf miner (Tuta absoluta) associated with organophosphates resistance. Journal Applied Entomology. 2017. https://doi.org/10.1111/jen.12386 ). Insecticides proved not to be a sustainable control method (1010. Biondi A, Guedes NRC, Wan F, Desneux N. Ecology, worldwide spread, and management of the invasive south american tomato pin- worm, Tuta absoluta: Past, present, and future. Annu Rev Entomol. 2018;63:239-258 https://doi.org/10.1146/annurevento-031616-034933 ) due to the pest resistance caused in some populations (1111. Siqueira HA, Guedes RN, Picanco MC. Insecticide resistance in populations of Tuta absoluta (Lepidoptera: Gelechiidae). Agricultural Entomology. 2000;2:147-153. , 1212. Biondi A, Zappalà L, Stark JD, Desneux N. Do Biopesticides Affect the Demographic Traits of a Parasitoid Wasp and Its Biocontrol Services through Sublethal Effects? PLoS ONE. 2013;9: e76548. https://doi.org/10.1371/journal.pone.0076548 ), as well as to high mortality of natural enemies of the pest (77. Bueno VHP, van Lenteren JC, Lins Jr, Calixto AM, Montes F, Silva D, et al. New records of Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) predation by Brazilian Hemipteran predatory bugs. Journal Applied Entomology. 2013;137:29-34. https://doi.org/10.1111/jen.12017 , 1313. Zappalà L, Siscaro G, Biondi A, Mollá O, González‐Cabrera J, Urbaneja A. Efficacy of sulphur on Tuta absoluta and its side effects on the predator Nesidiocoris tenuis. Journal of Applied Entomology. 2012;6:401-409. https://doi.org/10.1111/j.1439-0418.2011.01662.x ).
There is an increasing interest in mirid predators as biocontrol agents of T. absoluta and other tomato pests. Nesidiocoris tenuis (Reuter) and Macrolophus pygmaeus (Rambur) (Hemiptera: Miridae) are successfully marketed and used in the biological control of T. absoluta and Bemisia tabaci in Europe (1414. Calvo FJ, Lorente MJ, Stansly PA, Belda JE. Preplant release of Nesidiocoris tenuis and supplementary tactics for control of Tuta absoluta and Bemisia tabaci in greenhouse tomato. Entomologia Experimetalis et Applicata. 2012;143:111-119 https://doi.org/10.1111/j.1570-7458.2012.01238.x , 1515. van Lenteren JC, Bolckmans K, Köhl J, Ravensberg WJ, Urbaneja A. Biological control using invertebrates and microorganisms: plenty of new opportunities. BioControl. 2018a;63:39-59. https://doi.org/10.1007/s 10526-017-9801-4. ). Both M. pygmaeus and N. tenuis can prey 60-70 eggs and 2-3 larvae of first and second instar of TLM in 24 hours (1616. Urbaneja A, Monton H, Mollá O. Suitability of the tomato borer Tuta absoluta as prey for Macrolophus pygmaeus and Nesidiocoris tenuis. Journal Applied Entomology. 2009;133:292-296. https://doi.org/10.1111/j.1439-0418.2008.01319.x ). These predators coexist by feeding the same prey. Their interaction and the intraguild predation (IGP) were studied by Moreno-Ripoll et al. (1717. Moreno-Ripoll R, Agustí N, Berruezo R, Gabarra R. Conspecific and heterospecific interactions between two omnivorous predators on tomato. Biological Control. 2012;62:189-196. https://doi.org/10.1016/j.biocontrol.2012.04.005 ), Lampropoulos et al. (1818. Lampropoulos P, Perdikis D, Fantinou A. Are multiple predator effects directed by prey availability? Basic and Applied Ecology. 2013;14:605-613. https://doi.org/10.1016/j.baae.2013.08.004 ), and Perdikis et al. (1919. Perdikis D, Lucas E, Garantonakis N, Giatropoulos A, Kitsis P, Maselou D, et al. Intraguild predation and sublethal interactions between two zoophytophagous mirids, Macrolophus pygmaeus and Nesidiocoris tenuis. Biological Control. 2014;70:35-41. https://doi.org/10.1016/j.biocontrol.2013.12.003 ). However, this information is not available for other mirid species.
In Brazil, the mirid species Macrolophus basicornis (Stal) and Engytatus varians (Distant) were found in the tobacco (2020. Ferreira PS, da Silva E, Coelho LB. Miridae (Heteroptera) fitófagos e predadores de Minas Gerais, Brasil, com ênfase em espécies com potencial econômico. Iheringia, Série Zoologia. 2001;91:159-169.) and tomato crops (77. Bueno VHP, van Lenteren JC, Lins Jr, Calixto AM, Montes F, Silva D, et al. New records of Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) predation by Brazilian Hemipteran predatory bugs. Journal Applied Entomology. 2013;137:29-34. https://doi.org/10.1111/jen.12017 ). The results of these species provide elements to consider them as promising potential candidates for the biological control of T. absoluta (2121. Silva DB, Bueno VHP, Montes FC, van Lenteren JC. Population growth of three mirid predatory bugs feeding on eggs and larvae of Tuta absoluta on tomato. BioControl. 2016. https://doi.org/10.1007/s10526-016-9736-1 , 2222. van Lenteren JC, Hemerik L, Lins Jr and Bueno VHP. Functional Responses of Three Neotropical Mirid Predators to Eggs of Tuta absoluta on Tomato. Insects. 2016;7:34. https://doi.org/10.3390/insects7030034. , 2323. van Lenteren JC, Bueno VHP, Calvo FJ, Calixto AM, Montes FC. Comparative effectiveness and injury to tomato plants of three Neotropical mirid predators of Tuta absoluta (Lepidoptera: Gelechiidae). Journal of Economic Entomology. 2018b. https://doi.org/10.1093/jee/toy057 , 2424. van Lenteren JC, Bueno VHP, Burgio G, Lanzoni A, Montes FC, Silva D, et al. Pest kill rate as aggregate evaluation criterion to rank biological control agents:a case study with Neotropical predators of Tuta absoluta on tomato. Bulletin of Entomological Research. 2019. https://doi.org/10.1017/S0007485319000130 ). In laboratory conditions, M. basicornis and E. varians reached predation rates of 92-101 eggs of T. absoluta in 24 hours (77. Bueno VHP, van Lenteren JC, Lins Jr, Calixto AM, Montes F, Silva D, et al. New records of Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) predation by Brazilian Hemipteran predatory bugs. Journal Applied Entomology. 2013;137:29-34. https://doi.org/10.1111/jen.12017 , 2222. van Lenteren JC, Hemerik L, Lins Jr and Bueno VHP. Functional Responses of Three Neotropical Mirid Predators to Eggs of Tuta absoluta on Tomato. Insects. 2016;7:34. https://doi.org/10.3390/insects7030034. ). In addition, these mirids were able to prey eggs of Neoleucinodes elegantalis (Guenée) (Lepidoptera: Crambidae) known as “small tomato borer”, which co-occurs with T. absoluta in the tomato crop (2525. Martínez LD, Martínez Rivero MA, Bueno VHP, Collatz J. Predation behaviour and prey preference of two neotropical mirids against two key lepidopteran pests in tomato. International Journal of Tropical Insect Science 2021. https://doi.org/10.1007/s42690-021-00605-5 ). In the Caribbean, the presence of Macrolophus basicornis, E. varians, and N. tenuis showed a level of incidence higher than 50 % in Cuban crop systems such as tomato and tobacco (2626. Martínez MA, Duarte L, Baños HL, Rivas A, Sánchez A. Predatory mirids (Hemiptera: Heteroptera: Miridae) in tomato and tobacco in Cuba. Rev. Protección Veg. 2014;3:204-207).
In any agricultural system, it is difficult to determine the exact role of a given natural enemy of a pest in actually suppressing its host/prey population. Therefore, these effects need to be characterized and quantified, including possible key determinants on the prey population size (2727. Furlong MJ. Knowing your enemies: Integrating molecular and ecological methods to assess the impact of arthropod predators on crop pests. Insect Science. 2014;22:6-19. https://doi.org/10.1111/1744-7917.12157 ). In this sense, the studies related to the Multiple Predator Effects (MPEs) help to analyze the possible effects due to predators. In order to identify an emergent MPE, the observed impacts of multiple predators must be compared with an expected effect. These expectations depend on the knowledge of the system complexity. For example, for two predator types, A and B, the expected MPE is the sum of their individual effects; an emergent MPE is then defined as one that differs from this (2828. Sih A, Englund G, Wooster D. Emergent impacts of multiple predators on prey. Trends in Ecology & Evolution. 1998;13:350-355.).
The use of two types of models, additive and substitutive, can help to know multiple predator effects on predation rates (or prey mortality or survival) (2929. Soluk DA, Collins NC. Synergistic interactions between fish and stoneflies: Facilitation and interference among stream predators. Oikos. 1988;52:94-100., 2828. Sih A, Englund G, Wooster D. Emergent impacts of multiple predators on prey. Trends in Ecology & Evolution. 1998;13:350-355.). In the former, the additive design, compares the observed predation rates of individuals in each species with the predation rates of the predators when they are mixed. However, the additive expectation makes no sense when major impacts on prey by predators are expected and prey shortage is not prevented since it allows prey to be killed twice, which is unrealistic. To solve this overestimation of predation, a Multiplicative Risk Model (MRM) was proposed by Sih (2828. Sih A, Englund G, Wooster D. Emergent impacts of multiple predators on prey. Trends in Ecology & Evolution. 1998;13:350-355.). The second model, the substitutive design, relies on the total number of interacting individuals, which is constant while richness of the involved predator species changes (1818. Lampropoulos P, Perdikis D, Fantinou A. Are multiple predator effects directed by prey availability? Basic and Applied Ecology. 2013;14:605-613. https://doi.org/10.1016/j.baae.2013.08.004 ).
The aim of the present study was to define how the predation rates and the interaction of the mirid predators M. basicornis and E. varians behave when they fed on eggs and larvae (1st and 2nd instars) of T. absoluta. Predation rates of each mirid predators (alone or mixed) were determined and their effects, positive (increased prey consumption) or negative (decreased prey consumption, prey risk reduction), were assessed by using the MRM.
MATERIALS AND METHODS
⌅Insect rearing
⌅In order to have an available stock colony of the pest, tomato plants infested with eggs, larvae, and pupae of TLM were collected from cultivated areas in the campus of the Federal University of Lavras (Minas Gerais, Brazil). In the laboratory, the emerged adults were fed with tomato plants (cv. Santa Clara) in mesh cages (90 cm x 70 cm x 70 cm), where fresh tomato plants were regularly introduced to obtain individuals for the experiments.
Similarly, nymphs and adults of mirid predators were collected from tobacco plantations located in the municipalities of Ribeirão Vermelho (21°11′29′′S and 45°03′45′′W, at about 790 m altitude) and Lavras, (21°14′43′′S and 44°59′59′′W, at about 920 m altitude) Minas Gerais State, Brazil. The insects were taken to the laboratory and their stock colonies were reared in acrylic cages (60 x 30 x 30 cm). The predators were maintained on tobacco plants (Nicotiana tabacum L., cv TNN) and fed with eggs of Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) ad libitum, following the methodology described by Bueno et al. (77. Bueno VHP, van Lenteren JC, Lins Jr, Calixto AM, Montes F, Silva D, et al. New records of Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) predation by Brazilian Hemipteran predatory bugs. Journal Applied Entomology. 2013;137:29-34. https://doi.org/10.1111/jen.12017 ) and Silva et al. (2121. Silva DB, Bueno VHP, Montes FC, van Lenteren JC. Population growth of three mirid predatory bugs feeding on eggs and larvae of Tuta absoluta on tomato. BioControl. 2016. https://doi.org/10.1007/s10526-016-9736-1 ). The female mirids used in the experiments were taken from this stock colony with the aid of a compressor-vacuum pump equipment (FANEM®) used for sucking small insects.
All stock colonies were kept under the same environmental conditions in a room at 25±2oC, 60±10 % r.h., and 12 h photoperiod.
Experimental set up
⌅Predation rate of mirids on eggs and larvae of TLM: Females of each mirid species (1 to 5 days old) were individualized in 5 ml tubes containing a piece of moistened cotton (as water source) and kept starved for 24 hours. The tubes were sealed with a PVC® film to prevent them from escaping. The experimental unit made up by a uniform cohort of 120 TLM eggs was placed on tomato leaflets supported by a 1 % agar-water solution in a Petri dish (9 cm diameter). The eggs were carefully transferred to the tomato leaflets using a brush (Major Brushes® size 00). A film used to cover this experimental unit was pierced with an entomological needle to allow leaf transpiration and humidity removal from the dishes. One predator female was released into an experimental unit.
Similarly, the larvae of TLM were carefully transferred and maintained on tomato leaflets as described above. Ten and five larvae of 1st and 2nd instar were respectively placed in each experimental unit. Larval instars of T. absoluta was identified following the methodology described by Giustolin et al. (3030. Giustolini TA, Vendramin JD, Parra JRP. Número de instares larvais de Tuta absoluta (Meyrick) em genótipos de tomateiro. Scientiae Agricola. 2002;59:393-6. ). In the test, the number of larvae used was the same as used by Urbaneja et al. (1616. Urbaneja A, Monton H, Mollá O. Suitability of the tomato borer Tuta absoluta as prey for Macrolophus pygmaeus and Nesidiocoris tenuis. Journal Applied Entomology. 2009;133:292-296. https://doi.org/10.1111/j.1439-0418.2008.01319.x ) to determine the prey suitability of T. absoluta larval instars with N. tenuis and M. pygmaeus. However, given the smaller size and knowing the preference of both predators for 1st instar larvae, a double quantity of this instar was offered for M. basicornis and E. varians.
Eggs and larvae were exposed to predator females for 24 hours, and the number of preys consumed was then counted under a stereomicroscope (Zeiss 40X). The prey was considered as consumed when the eggshell was present without content and the larvae were immobile and without abdominal content. The experiments on different types of prey were carried out at 25±1ºC, 70±10 % r.h., and 12h photoperiod in a climatic chamber ELECTROlab®.
The following experimental treatments were set up: (1) one M. basicornis female offered with TLM eggs (MB_alone), (2) one M. basicornis female with first instar larvae of TLM (MB_alone), (3) one M. basicornis female with second instar larvae of TLM (MB_alone), (4) one E. varians female with TLM eggs (EV_alone), (5) one E. varians female with first instar larvae of TLM (EV_alone), (6) one E. varians female with second instar larvae of TLM (EV_alone). One female of both species (M. basicornis + E. varians) with prey (i. e. (7) (MB+EV_together) with TLM eggs, (8) (MB+EV_together) with first instar larvae and (9) (MB+EV_together) with second instar larvae). Twenty-five replicates per treatment were assayed.
Multiple predation effects in interspecific interactions: Predation by the combination M. basicornis and E. varians feeding on eggs and larvae of TLM was determined and predicted. A Multiplicative Risk Model (MRM) was calculated following the methodologies proposed by Soluk (3131. Soluk DA. Multiple prey effects: Predicting mixed functional response of stream fish and invertebrate predators. Ecology. 1993;74:19-225.); Soluk and Collins (2929. Soluk DA, Collins NC. Synergistic interactions between fish and stoneflies: Facilitation and interference among stream predators. Oikos. 1988;52:94-100.); Sih et al. (2828. Sih A, Englund G, Wooster D. Emergent impacts of multiple predators on prey. Trends in Ecology & Evolution. 1998;13:350-355.); Griffen (3232. Griffen B. Detecting emergent effects of multiple predator species. Oecologia. 2006;148:702-709. https://doi.org/10.1007/s00442-006-0414-3 ), Schmitz (3333. Schmitz OJ. Predator diversity and trophic interactions. Ecology. 2007;88:2415-2426), and Lampropoulos et al. (1818. Lampropoulos P, Perdikis D, Fantinou A. Are multiple predator effects directed by prey availability? Basic and Applied Ecology. 2013;14:605-613. https://doi.org/10.1016/j.baae.2013.08.004 ). According to these authors, this model applies to the additive design and it is considered to account for prey removal because an individual prey eaten by one of the species is no longer available to the other. If predators have independent effects, then the proportion of the prey that survives both predators will be:
Where Pa and Pb is the prey mortality when exposed to predator a or b singly.
The predation observed when both predators were present was estimated by
Where Cfs is the expected mixed consumption, Np is the initial prey density, and Pa and Pb as described above over 24 hours of exposure.
Statistical analysis
⌅Normal distribution of data was corroborated by the Shapiro-Wilk test for all the experiments. The effect of treatments the predation rate (in per cent) during 24 h was analyzed using a two-way ANOVA with predators as the first factor with three levels: each predator released individually and mixed. The second factor, also with three levels, was type of prey: eggs and larvae of 1st and 2nd instar, respectively. In addition, the interaction between factors was included in the model. Taking into account the significant differences obtained for interaction term, the predation rates reached by predator combinations between treatments was compared for each type of prey separately using a one-way ANOVA test. The comparisons between means were performed using Tukey HSD post-hoc tests (p<0.05).
The comparisons between predation rates observed and predicted from the MRM to test emergent multiple predator effects (MPEs) with M. basicornis and E. varians mixed were analyzed by a two-way ANOVA followed by two factors: a) type of data, observed versus predicted predation rates, and b) type of prey (i.e. eggs and 1st and 2nd instar larvae). In addition, the interaction between factors was included in the model. Data used in the analysis were log transformed. The comparisons between means were performed by Tukey HSD post-hoc test (p<0.05). The statistical analyses were carried out with R v.3.6.3 (3434. R Core Team R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. 2018. URL https://www.R-project.org/ ). The standard error for all mean values (mean ± SE) was provided.
RESULTS
⌅Predation rate of mirids on eggs and larvae of TLM
⌅The predation rate of M. basicornis and E. varians on eggs and larvae of T. absoluta significantly differed among predator combinations (F 2, 216 = 19.53, P < 0.0001), type of prey (F 2, 216 = 205.50, P < 0.0001), and prey-predator interaction (F 4, 216 = 11.99, P < 0.0001). Both predators individually reached similar predation rates on eggs with no significant differences; however, their combination significantly increased their eggs consumption (for M. basicornis: F 2, 72 = 14.37, P < 0.001 and for E. varians: F 2, 72 = 14.37, P < 0.0001) (Fig. 1).
Feeding on firstt instar larvae of T. absoluta by each mirid species individually differed significantly (F 2, 72 = 18.39, P < 0.0001). Macrolophus basicornis reached the highest predation rate of, which did not differ from that observed when both predators were present and in contrast with the response obtained by E. varians (F 2, 72 = 18.39, P < 0.0001). However, predation on the second instar larvae of the pest did not show significant differences between treatments (F 2, 72 = 3.09, P = 0.051), but the adjustment of the p-values showed no differences between the predator combinations on this type of prey (Fig. 1).
Interspecific interaction effects on the predation efficacy
⌅Differences between observed and predicted predation rates were shown by the MRM (OP-rates) and type of preys (TP) (Table 1.). No interaction between the factors was shown by their analysis using the two-way ANOVA.
| Sourse | df | SS | F | P |
|---|---|---|---|---|
| OP-rates | 1 | 0.201 | 7.125 | 0.008 |
| TP | 2 | 11.964 | 212.047 | <.0001 |
| OP-rates x TP | 2 | 0.158 | 2.792 | 0.064 |
| Residuals | 144 |
The analysis of multiple predator effects (MPEs) indicated that when both mirid predators were present feeding on eggs (P = 0.987) or lst instar larvae (P = 0.995), the results were positive because of the similarity between the observed and predicted values estimated by the model (MRM), without significant differences (Fig. 2). In contrast, the effect of both mirids feeding on 2nd instar larvae was negative, as the observed predation rate was significantly lower than the estimated by the MRM (P = 0.009) (Fig. 2).
DISCUSSION
⌅The efficacy of the two neotropical mirids on eggs and larvae of the pest is known (2121. Silva DB, Bueno VHP, Montes FC, van Lenteren JC. Population growth of three mirid predatory bugs feeding on eggs and larvae of Tuta absoluta on tomato. BioControl. 2016. https://doi.org/10.1007/s10526-016-9736-1 , 2424. van Lenteren JC, Bueno VHP, Burgio G, Lanzoni A, Montes FC, Silva D, et al. Pest kill rate as aggregate evaluation criterion to rank biological control agents:a case study with Neotropical predators of Tuta absoluta on tomato. Bulletin of Entomological Research. 2019. https://doi.org/10.1017/S0007485319000130 ). However, our results show their preference for eggs and the 1st instar larvae, in contrast with what was observed for the 2nd instar. Our results are in agreement with those observed by Urbaneja et al. (3535. Urbaneja A, González-Cabrera J, Arnó J, Gabarra R. Prospects for the biological control of Tuta absoluta in tomatoes of the Mediterranean basin. Pest Management Science. 2012;68:1215-1222. https://doi.org/10.1002/ps.3344. ) with other mirid species such as N. tenuis and M. pygmaeus .
The positive response of the two mirid species used together against eggs and first instar larvae of TLM, and their negative response on second instar larvae is related to main mechanisms of ‘multiple predator effects’ (MPEs). According to Sih et al. (2828. Sih A, Englund G, Wooster D. Emergent impacts of multiple predators on prey. Trends in Ecology & Evolution. 1998;13:350-355.) the negative response occurs when the prey defense effects against one predator are greater than against the other one.
Interactions between predators (e.g., cooperation, competition or intraguild predation), as well as antipredator responses by prey, can lead to emergent MPEs, where prey consumption rates by multiple predators foraging together cannot be predicted by knowing the independent effects of each predator on prey survival (3636. McCoy W, Stier C, Osenberg W. Emergent effects of multiple predators on prey survival: the importance of depletion and the functional response. Ecology Letters. 2012;15:1449- 1456. https://doi.org/10.1111/ele.12005 ). Predator-prey interactions are known to be an important factor in shaping populations of organisms in nature, including the size, type, and dispersion altering the intensity of herbivore-plant interactions (3737. Tabic A, Yonah R, Coll M. Association between omnivorous Orius bugs and their thrips prey at different spatial scales of Verbesina encelioides flowers. Israel Journal of Plant Sciences. 2010;58:131-141. https://doi.org/10.1560/IJPS.58.2.131 ). Predators may induce an antipredator behavior of prey that affects feeding and dispersal of mobile prey (3838. Belliure B, Amorós-Jiménez R, Fereres A, Marcos-García MA. Antipredator behaviour of Myzus persicae affects transmission efficiency of Broad bean wiltvirus 1. Virus Research. 2011;159:206-214. , 3939. Duarte L, Pacheco R, Quiñones M, Martínez MA, Bueno VHP. Nesidiocoris tenuis Reuter (Hemiptera: Miridae) and Cycloneda sanguinea limbifer (Casey) (Coleoptera: Coccinelidae): Behaviour and predatory activity on Myzus persicae Zulzer (Hemiptera: Aphididae). Revi. Protección Veg. 2014;29:99-105 ).
In order to explain the reaction of TLM larvae on predation rate of mirids, behavior of the predators and prey during the experiment was observed. The larvae of T. absoluta showed an escape and defense behavior after the first contact with the predators and reacted against the predators with strong movements (first and second instars) and evasion. In addition, when M. basicornis and E. varians were mixed for feeding on larvae, the number of encounters between both predators was very rare. After their interaction, they evaded each other by moving in opposite directions. Sometimes, E. varians remained immobile compared to M. basicornis. These observations suggest that E. varians perceives the presence of another predator species and reacts by displaying this pattern. Olson et al. (4040. Olson RS, Hintze A, Dyer FC, Knoester DB, Adami C. Predator confusion is sufficient to evolve swarming behaviour. Journal of the Royal Society Interface. 2013;10:0305. https://doi.org/10.1098/rsif.2013.0305 ) evidenced that predatory confusion or a relatively simple limited perception could have persistent evolutionary effects on prey behavior, sensorial mechanisms of the predators, and interaction between them.
The tests involving TLM larvae as prey suggest an interspecific predator interaction with an additional prey-predator interaction, as motile larvae are more likely to escape predation. This interference showed a reducing effect on the predation rate, which was more evident with second instar larvae as prey. These larvae are larger and can vigorously defend themselves.
However, the reaction of the encounter between predators could stimulate the consumption of eggs and first instar larvae because the mirids would need to move and cover a major area on the leaflets, with higher possibilities for encountering intact prey. Perdikis et al. (1919. Perdikis D, Lucas E, Garantonakis N, Giatropoulos A, Kitsis P, Maselou D, et al. Intraguild predation and sublethal interactions between two zoophytophagous mirids, Macrolophus pygmaeus and Nesidiocoris tenuis. Biological Control. 2014;70:35-41. https://doi.org/10.1016/j.biocontrol.2013.12.003 ) did not observe aggressive contacts between the predators when they studied the behavioral activities of M. pygmaeus and N. tenuis,, but N. tenuis was affected by the presence of M. pygmaeus. A similar result was obtained with M. basicornis and E. varians in this study, where an aggressive behavior was not observed.
In the present study, although the combined use of both predators increased consumption of eggs and first instar larvae of the pest, no significantly for M. basicornis in the latter case, and revealed the potential of both predators, the implications of their coexistence in crop areas are still in discussion. In this sense, Tylianakis et al. (4141. Tylianakis JM, Rand TA, Kahmen A, Klein AM, Buchmann N, Perner J, et al. Resource heterogeneity moderates the biodiversity-function relationship in real world ecosystems. PLoS Biology. 2008;6(5):e122. https://doi.org/10.1371/journal.pbio.0060122. ) and Tylianakis and Romo (4242. Tylianakis JM, Romo CM. Natural enemy diversity and biological control: Making sense of the context-dependency. Basic and Applied Ecology. 2010;11:657-668. https://doi.org/10.1016/j.baae.2010.08.005. ) suggested that more complex habitats could increase the positive effects of multiple predators; for this reason, it would be very interesting to find out whether the effect of the presence of individuals of these two predator species in the field is complementary or competitive on prey regulation. In the context of biological control, the coexistence of natural enemies and the role they play by competition are very important. Indeed, niche competitions have been considered a key mechanism associated with the prey regulation by predator biodiversity (4343. Cusumano A, Peri E, Vinson SB, Colazza S. Intraguild interactions between two egg parasitoids exploring host patches. BioControl. 2011;56:173-184. https://doi.org/10.1007/s10526-010-9320-z. , 4444. Rondoni G, Onofri A, Ricci C. Differential susceptibility in a specialized aphidophagous ladybird, Platynaspisluteo rubra (Coleoptera: Coccinellidae), facing intraguild predation by exotic and native generalist predators. Biocontrol Science and Technology. 2012;2211:1334-1350. https://doi.org/10.1080/09583157.2012.726607 ).
The density dependent effects of a single species is as important as those of multiple species. Especially given by the fact that many predators occupy a specific niche on a plant or crop (1919. Perdikis D, Lucas E, Garantonakis N, Giatropoulos A, Kitsis P, Maselou D, et al. Intraguild predation and sublethal interactions between two zoophytophagous mirids, Macrolophus pygmaeus and Nesidiocoris tenuis. Biological Control. 2014;70:35-41. https://doi.org/10.1016/j.biocontrol.2013.12.003 ). However, our first findings on how these predators when used together may react to control T. absoluta under laboratory conditions are the basis for future research, as both predators are being widely studied as potential biocontrol agents of this target and other pests in tomato (2525. Martínez LD, Martínez Rivero MA, Bueno VHP, Collatz J. Predation behaviour and prey preference of two neotropical mirids against two key lepidopteran pests in tomato. International Journal of Tropical Insect Science 2021. https://doi.org/10.1007/s42690-021-00605-5 ) and their preference for a specific niche could change and share the same plant stratum under different temperature scenarios (Author's personal communication).
On the other hand, intraguild predation (IGP) occurs when a predatory species attacks other competing species (4545. Gagnon AE, Heimpel GE, Brodeur J. The Ubiquity of Intraguild Predation among Predatory 474 Arthropods. PLoS ONE. 2011;11:e28061. 475 https://doi.org/10.1371/journal.pone.0028061 ). Therefore, the potential implications of our results should be considered for biological control. Further studies are imperative to understand IGP interactions between E. varians and M. basicornis and their implications in the regulation of T. absoluta as it is known for other mirid predators of this prey. For example, M. pygmaeus nymphs completed their development to the adult stage only when the alternative prey (eggs of Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) was present, while in the presence of N. tenuis adults, a greater number of dead M. pygmaeus nymphswere observed with their body fluids totally sucked out, indicating an interaction of intraguild predation (1919. Perdikis D, Lucas E, Garantonakis N, Giatropoulos A, Kitsis P, Maselou D, et al. Intraguild predation and sublethal interactions between two zoophytophagous mirids, Macrolophus pygmaeus and Nesidiocoris tenuis. Biological Control. 2014;70:35-41. https://doi.org/10.1016/j.biocontrol.2013.12.003 ).
CONCLUSION
⌅Our findings showed the efficacy of E. varians and M. basicornis simultaneously on T. absoluta as prey, where the predators achieved suppression of its immobile and mobile stages (i.e., eggs and larvae) alone or mixed. According to the MRM, the mirids in combination showed a positive (additive) effect when preying on eggs and first instar larvae, but a negative effect on second instar larvae. Although M. basicornis has a greater predatory capacity compared to E. varians on first instar larvae of the pest, both mirids are good candidates as biocontrol agents against T. absoluta on tomato.