Introduction
Generalist predators contribute to the management of a wide range of agricultural pests, around the world (Symondson et al., Reference Symondson, Sunderland and Greenstone2002; Bouvet et al., Reference Bouvet, Urbaneja, Pérez-Hedo and Monzó2019). In Europe, several integrated pest management (IPM) programmes, based on the augmentation and conservation of polyphagous predators, have been successfully developed and implemented (Pérez-Hedo and Urbaneja, Reference Pérez-Hedo, Urbaneja, Horowitz and Ishaaya2016; van Lenteren et al., Reference van Lenteren, Bolckmans, Köhl, Ravensberg and Urbaneja2018, Reference van Lenteren, Alomar, Ravensberg, Urbaneja, Gullino, Albajes and Nicot2020). Releases of predatory mirid bugs (Hemiptera: Miridae) [Nesidiocoris tenuis Reuter in Southern Europe and Macrolophus pygmaeus (Rambur) in Northern Europe] and conservation of their natural populations have resulted in effective control of key tomato (Solanum lycopersicum L.) pests, including the sweet potato whitefly Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) and the South American tomato pinworm Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) (Arnó et al., Reference Arnó, Gabarra, Liu, Simmons, Gerling, Stansly and Naranjo2010; Urbaneja et al., Reference Urbaneja, González-Cabrera, Arnó and Gabarra2012; Pérez-Hedo et al., Reference Pérez-Hedo, Suay, Alonso, Ruocco, Giorgini, Poncet and Urbaneja2017; van Lenteren et al., Reference van Lenteren, Alomar, Ravensberg, Urbaneja, Gullino, Albajes and Nicot2020). However, zoophytophagous mirids can cause damage to crop plants when prey is scarce or they are confined in greenhouses (Pérez-Hedo et al., Reference Pérez-Hedo, Riahi and Urbaneja2021; Chinchilla-Ramírez et al., Reference Chinchilla-Ramírez, Garzo, Fereres, Gavara-Vidal, ten Broeke, van Loon and Pérez-Hedo2021).
Following the success achieved with the use of predatory mirids in Europe horticultural crops (van Lenteren et al., Reference van Lenteren, Bolckmans, Köhl, Ravensberg and Urbaneja2018, Reference van Lenteren, Alomar, Ravensberg, Urbaneja, Gullino, Albajes and Nicot2020), efforts to find a native species within this group of predators on the American continent have increased (Bueno et al., Reference Bueno, van Lenteren, Lins, Calixto, Montes, Silva and Pérez2013, Reference Bueno, Montes, Sampaio, Calixto and van Lenteren2018; Lellani et al., Reference Lellani, Díaz, Li, Li, Martínez, Rivero and Bueno2014; Silva et al., Reference Silva, Bueno, Montes and van Lenteren2016; Smith and Krey, Reference Smith and Krey2019; van Lenteren et al., Reference van Lenteren, Bueno, Burgio, Lanzoni, Montes, Silva and Hemerik2019; Pérez-Hedo et al., Reference Pérez-Hedo, Riahi and Urbaneja2021; Roda et al., Reference Roda, Castillo, Allen, Urbaneja, Pérez-Hedo, Weihman and Stansly2020). The reduced efficacy of IPM programmes due to insecticide resistance acquired by B. tabaci (Stansly and Naranjo, Reference Stansly and Naranjo2010) and the threat of multiple invasive pests, such as T. absoluta and the old World bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in North America (Biondi and Desneux, Reference Biondi and Desneux2019; Tembrock et al., Reference Tembrock, Timm, Zink and Gilligan2019), has compelled this search for management alternatives. Native mirid species may provide similar control as known biological agents with the added advantages of being already adapted to local conditions, and they would not require quarantine evaluation and release permits (Shipp and Wang, Reference Shipp and Wang2006; Calvo et al., Reference Calvo, Torres-Ruiz, Velázquez-González, Rodríguez-Leyva and Lomeli-Flores2016; Pérez-Hedo et al., Reference Pérez-Hedo, Riahi and Urbaneja2021).
Macrolophus praeclarus (Distant) (Hemiptera: Miridae) has been previously reported in Central and South Florida and Puerto Rico, and several tropical countries of the American continent including Brazil, Colombia, Cuba, Dominic Republic, French Guyana, Guatemala, Mexico, Nicaragua, Paraguay, Peru, and Suriname (Carvalho and Rosas, Reference Carvalho and Rosas1965; Carvalho et al., Reference Carvalho, Da, Afonso, Paulo and Roberto1977; Cassis, Reference Cassis1984; Chérot et al., Reference Chérot, Carpintero and Maes2007; Ferreira and Henry, Reference Ferreira and Henry2011; Martínez et al., Reference Martínez, Duarte, Baños, Rivas and Sánchez2014; Chérot and Carpintero, Reference Chérot and Carpintero2016; Serra and van Lenteren, Reference Serra, van Lenteren, van Lenteren, Bueno, Luna and Colmeneraz2020). In these countries, M. praeclarus has been found mainly on tobacco (Nicotiana tabacum L.) and tomato crops, although it has also been reported on citrus and its associated weeds (Arellano and Vergara, Reference Arellano and Vergara2016), on non-cultivated plants such as Martynia annua L. (Lamiales: Martyniaceae) (Vera and Ayala, Reference Vera and Ayala1979) and succulent sesame [(Uncarina grandidieri (Baill.) Stapf (Lamiales: Pedaliaceae)] (Roda et al., Reference Roda, Castillo, Allen, Urbaneja, Pérez-Hedo, Weihman and Stansly2020). Like other mirids, M. praeclarus is a polyphagous predator, preying on eggs and early instars of the tobacco budworm Heliothis virescens (Fab.) (Lepidoptera: Noctuidae), green peach aphid Myzus persicae (Sulzer) (Hemiptera: Aphididae), onion thrips Thrips tabaci Lindeman (Thysanoptera: Thripidae) and eggs and early instars of B. tabaci (Ayala et al., Reference Ayala, Grillo and Vera1982; Soto and Nakano, Reference Soto and Nakano2009; Roda et al., Reference Roda, Castillo, Allen, Urbaneja, Pérez-Hedo, Weihman and Stansly2020).
Recent studies have shown the ability of M. praeclarus to control B. tabaci in field cage studies under South Florida spring and fall conditions (Roda et al., Reference Roda, Castillo, Allen, Urbaneja, Pérez-Hedo, Weihman and Stansly2020). Ten days after infesting tomato plants with 10 adult whiteflies, the release of M. praeclarus (1 male and 1 female/plant) reduced the total number of whiteflies on infested tomatoes by close to 100% 6 weeks after adding the mirids. In fact, M. praeclarus reduced the number of whitefly nymphs earlier than N. tenuis. Macrolophus praeclarus was able to increase its population levels in tomato plants, reaching levels around 30 individuals/plant, without causing plant damage. In contrast, N. tenuis and Engytatus modestus (Distant) (Hemiptera: Miridae) cause damage to tomato plants under the same experimental conditions (Roda et al., Reference Roda, Castillo, Allen, Urbaneja, Pérez-Hedo, Weihman and Stansly2020). Despite these promising results, the biology of M. praeclarus is almost unknown. We characterized the South Florida population of M. praeclarus' molecularly (Supplementary Material) and determined M. praeclarus' developmental thermal requirements, its predation capacity at five constant temperatures, and its host plant odour preference for tomato, sweet pepper and eggplant. Finally, we evaluated, under controlled conditions, the potential of this species to cause plant damage on tomato and assessed whether its phytophagy can induce defensive plant responses in tomato plants. This information will help to clarify the region and crops where M. praeclarus could be used as a biological control agent, to help in the development of mass rearing techniques, and to guide subsequent studies for its field application.
Materials and methods
Plants and insects
Macrolophus praeclarus was collected from ornamental succulent sesame located at Fairchild Botanical Gardens in Miami, Florida, USA. This population of M. praeclarus was characterized by molecular methods and the accession number MT154517 was submitted to the GenBank (Supplementary Material). Nesidiocoris tenuis was collected from the same plant species but at a private residence in Miami Beach, Florida. These field collected mirids were used to establish colonies on pesticide-free tomato seedling var. Sweet ‘n’ Neat Cherry (Bonnie Plants; Home Depot, Fort Myers, FL) at University of Florida, Southwest Florida Research and Education Center (SWFREC), Immokalee, FL. A mix of frozen eggs of Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) and cysts of the crustacean Artemia spp. (Anostraca: Artemiidae) (1:5 w:w) (Entofood®; Koppert B.S.) was used as an artificial source of prey for the mirids in the colonies. Only frozen eggs of E. kuehniella (supplied by Koppert B.S.) were used in the experiments described below.
Tomato var. Grand Marshall, sweet pepper (Capsicum annuum L.) var. Antebellum and eggplant (Solanum melongena L.) var. Black Beauty (Barnett, Parting Plants, Inc. Immokalee, FL) plants were transplanted into individual 0.5-L pots. Plants were not fertilized or treated with pesticides. Colonies and plants were maintained at 25 ± 1°C, 60% humidity and 14:10 h (L:D) photoperiodic conditions in climatic chambers at SWFREC.
Development and survival of M. praeclarus at different temperatures
The developmental time from egg to adult and nymphal survival of M. praeclarus was studied at five constant temperatures (20, 25, 30, 33 and 35°C) with a 14:10 h photoperiod and 70 ± 10% relative humidity. Macrolophus praeclarus oviposition was synchronized in order to obtain a similarly aged cohort for each temperature. For each experimental temperature, 30 pairs of M. praeclarus were introduced inside a plastic cage (60 × 60 × 60 cm, BugDorm-2 insect tents; MegaView Science Co., Ltd.; Taichung, Taiwan) containing two clean tomato plants (~20 cm). After 24 h, the main and secondary stems of the middle-apical part of each of the plants, which contained eggs less than 24 h old, were cut into sections approximately 5 cm long. The stem pieces were place into Petri dishes (140 mm diameter) and held in Percival incubators (Model I-36LL-1, Percival Scientific Inc. Perry, IA, USA) at each test temperature. A ball of wet cotton was added to maintain humidity inside each Petri dish. The Petri dishes were checked daily for 1st instar nymphs less than 24 h after emergence. The egg developmental time for each nymph was considered as the period from the cohort formation until the detection of the nymph. These nymphs were transferred individually to Petri dishes (45 mm diameter) that contained a 2 cm tomato stem piece, a moistened piece of cotton, and E. kuehniella eggs provided ad libitum. Daily, the instar of each individual nymph was checked and the tomato stem piece replaced, until the adult stage was recorded or the nymph was found dead. Changes from one nymphal stage to another were recorded when they molted and exuvia was observed.
Temperature thresholds and thermal constant
Total developmental rates (egg-adult) (y = 1/developmental time) were plotted against temperatures and fitted with Lactin model for nonlinear regression (Lactin et al., Reference Lactin, Holliday, Johnson and Craigen1995): r(T) = 
${\rm e}^{\rho T}-{\rm e}^{( \rho T_{\max }-T{}_{\max }-T) /\Delta ) } + \lambda $, where r(T) is the developmental rate at temperature T, T max is the temperature of maximal developmental rate, ρ, Δ and λ are fitted parameters. The upper developmental threshold and maximal developmental rate were estimated from the regression. Developmental rates for temperatures below T max were fitted with a linear regression (y = a + bT). This was required to estimate the lower developmental threshold (T = −a/b) and the heat accumulation in degree-days (K = 100/b) required to complete each stage above the threshold (T) (Campbell et al., Reference Campbell, Frazer, Gilbert, Gutierrez and Mackauer1974). The standard error of K was the standard error of the slope (b) divided by the square of the slope.
Predation capacity of M. praeclarus at different temperatures
The capacity of female and male M. praeclarus to feed on E. kuehniella eggs was evaluated in a no-choice experiment under five constant temperatures (20, 25, 30, 33 and 35°C) with a 14:10 h photoperiod and 70 ± 10% relative humidity. Female and male M. praeclarus, randomly selected from the colony, were individually placed inside a 5 ml vial and starved for 8 h before use. Water was provided by placing a piece of saturated cotton in the vial. After the starvation period, individuals were introduced individually into experimental arena, a 45 mm diameter Petri dish with 120 E. kuehniella eggs. Eggs were placed on the sticky surface of a post-it note (Post-it® Notes, 3 M, Cynthiana, KY, USA). In each experimental arena, a ball of wet cotton was added as a water source for the mirid and to maintain humidity inside the Petri dish. After 24 h, the predators were removed from the arenas and the number of eggs preyed upon was counted for the 5 temperatures using a dissecting microscope (10×). The presence of empty chorions was used as evidence of predation (Mollá et al., Reference Mollá, Biondi, Alonso-Valiente and Urbaneja2014). At least 15 replicates were performed for each temperature, except for 33°C where only nine replicates were obtained.
Preference of M. praeclarus for sweet pepper, eggplant and tomato odours
A Y-tube olfactometer experiment was conducted to test the olfactory responses of M. praeclarus females to sweet pepper, eggplant and tomato. The females were less than 7 days old, presumptively mated, and were starved for 24 h. The Y-tube olfactometer (Analytical Research Systems, Gainesville, FL) consisted of a 2.4-cm-diameter Y-shaped glass tube with a 13.5-cm long base and two arms each 5.75 cm long (Pérez-Hedo and Urbaneja, Reference Pérez-Hedo and Urbaneja2015). Both side arms were connected via high-density polyethylene (HDPE) tubes to two identical glass jars (2.5 l volume). Each jar was connected to an air pump that produced a unidirectional humidified airflow at 150 ml min−1. Three 60-cm T8 LED integrated tubes (EHI48-T822, Energy Harness Corporation (EHC), Cape Coral, FL) were positioned 40 cm above the olfactometer. All Y-tube experiments were conducted in climatic chambers (23 ± 2°C, 60 ± 10% RH). The tomato, eggplant and pepper plants, approximately 20 cm in height, were placed in the jars 10 min before adding a single female into the entry of the tube. The mirid was observed until she had walked at least 3 cm up one of the arms or until 15 min had elapsed. Females that did not select a side arm within 15 min were recorded as ‘no-choice’ and were excluded from data analysis. A minimum of 33 mirids that selected an odour source were recorded for each pair. After recording responses of five mirids, the Y-tube was washed with soap and water, rinsed with acetone, and allowed to dry for 5 min. The test plants were subsequently switched between the left and right side arms to minimize any spatial effect on choice. One plant was used to test the response of 10 females and they were replaced with new plants for the remaining replicates.
Phytophagy by M. praeclarus
Phytophagy by M. praeclarus was evaluated by comparing it with N. tenuis, a species known to cause damage under these experimental conditions (Pérez-Hedo and Urbaneja, Reference Pérez-Hedo, Urbaneja, Horowitz and Ishaaya2016; Chinchilla-Ramírez et al., Reference Chinchilla-Ramírez, Garzo, Fereres, Gavara-Vidal, ten Broeke, van Loon and Pérez-Hedo2021). The number of necrotic rings caused by mirid feeding were counted as well as the number of withered leaflets produced by 10 adults (five males and five females) on a fully expanded young leaf of the apical part of an entire tomato plant (Urbaneja-Bernat et al., Reference Urbaneja-Bernat, Bru, González-Cabrera, Urbaneja and Tena2019; Chinchilla-Ramírez et al., Reference Chinchilla-Ramírez, Pérez-Hedo, Pannebakker and Urbaneja2020). The insects used had completed their final molt 3−5 days prior to the start of the experiment. The plants were held in a Percival incubator at 25°C, 60% humidity and 14:10 h (L:D) photoperiod and watered every 2 days. Each leaf was enclosed in a muslin bag (15 × 21 cm) with 10 adults for 5 days without a supplementary food source. The dead individuals were removed daily from muslin bags to prevent necrophagy during this experiment. At the end of the experiment, all remaining insects were counted and total mortality was recorded. Phytophagy was evaluated by counting the number of necrotic rings visible on the petiole and the rachis. The number of withered leaflets per leaf was counted as indicator of wilting. Eight replicates were performed for each species.
Plant defense response induction by M. praeclarus
To assess the induction potential of plant defensive responses by the mirid feeding on tomato plants, 20 individuals (sex ratio 1:1) were released into a cage (60 × 60 × 60 cm BugDorm-2 insect tents) containing a single tomato plant (25 cm) and allowed to feed. After 24 h, the mirids were removed and the apical part of the plant was cut and immediately ground in liquid nitrogen. The same amount of material was collected from clean plants that had not been in contact with any arthropod since germination. Six replicates were performed per treatment. For each replicate, 100 mg of the samples were used for RNA extraction to determine the expression levels of genes related to plant defense. Specifically, ASR1 (marker gene for abscisic acid, ABA), PIN2 (marker gene for jasmonic acid, JA) and PR1 (marker gene for the salicylic acid signalling pathway, SA) were quantified. Total RNA was extracted using a plant RNA extraction kit (Omega Bio-Tek Inc., Doraville, GA, USA), which was treated with DNase-free DNase (Promega Corporation, Madison, Wisconsin, USA) to eliminate genomic DNA contamination. The RT reaction and the SYBR PCR reaction was performed as described by Pérez-Hedo et al. (Reference Pérez-Hedo, Bouagga, Jaques, Flors and Urbaneja2015). Quantitative PCR was performed using the Smart Cycler II sequence detector (Cepheid, Sunnyvale, CA, USA) with standard PCR conditions. Expression of EF1 (elongation factor-1) was used as a standard control gene for normalization. The nucleotide sequences of the gene-specific primers are described in table 1.
Table 1. Primers used for quantification of EF1 (elongation factor-1), ASR1 (abscisic acid stress ripening protein), PR1 (pathogenesis-related protein 1) and PIN2 (JA-regulated defense protein) genes.
Data analysis
The data from the olfactory responses were analysed using a chi-squared goodness-of-fit test based on a null model where the odour sources were selected with equal frequency. Predation capacity and developmental time were subjected to two-way analysis of variance to evaluate the effect of factors sex and temperature, using Tukey's test for mean separation in the event of a significant F (P < 0.05). To determine whether survival and sex ratio of M. praeclarus was affected by temperature, the fitted values of the expected sex ratio and of survival were obtained with a generalized linear model (GLM) with quasibinomial family selected as the appropriate model. The mirid mortality in the phytophagy experiment and the gene expression analyses were analysed using one-tailed Student's t test (P ˂ 0.05). All analysis were performed with SPSS software (IBM SPSS, 2004) except the GLM which was conducted with the R freeware statistical package (Version 1.0.143) (RStudio Team, 2015) and the fit of the Lactin nonlinear model using a least-squares method which was established with TableCurve 2D program (Systat Software Inc., San José, California).
Results
Development and survival of M. praeclarus at different temperatures
Macrolophus praeclarus completed development from egg to adult at 20, 25, 30 and 33°C (table 2). However, individuals tested at 35°C did not survive beyond the second nymphal instar. The duration of development did not differ between sexes (F = 0.338; df = 1, 102 P = 0.562), and developmental times were grouped (table 2). The developmental time at 30 and 33°C was significantly shorter than that found at 25°C, which was shorter than that at 20°C (F = 1237.6; df = 3, 102 P < 0.0001). No interaction between temperature and sex was found (F = 0.300; df = 3, 102 P < 0.825).
Table 2. Mean developmental times (days ± SE) for eggs and nymphs, nymphal survival (%) and sex ratio (females/total) of M. praeclarus when reared on E. kuehniella eggs on detached tomato stem pieces at five constant temperatures and 14:10 h L:D.
Male and female development times were grouped. For total developmental time, data followed by a different letter are significantly different (P < 0.05, Tukey test).
The survival of M. praeclarus decreased with increasing temperature (GLM quasibinomial: χ2 = 230.83; P < 0.0001) (fig. S2). At 20°C, 78.0% of the individuals completed their cycle and only 39.0% completed it at 33°C. Although, the sex ratio of M. praeclarus was biased towards females at 20°C and towards males at 33°C, there was no significant effect of temperature on the sex ratio of this species (GLM quasibinomial: χ2 = 2.165; P = 0.539).
Temperature thresholds, and thermal constant
Rates of development (y) were fitted with a nonlinear regression [
$r( T) = {\rm e}^{\rho T}-{\rm e}^{( \rho T_{\max }-T{}_{\max }-T) /\Delta ) } + \lambda $] (ρ = 0.0598 ± 0.0017, Δ = 1.3151 ± 0.4528, λ = −1.4222 ± 0.0037; estimate ± SE)] (r 2 = 0.9597; F = 785.9; df = 3, 134; P < 0.0001). The upper developmental threshold was 35.3°C, and maximal developmental rate occurred at 31.7°C (fig. 1). For estimating lower temperature thresholds, developmental rates observed between 20° and 33°C were further fitted with a linear regression (y = 0.2204x–2.4762; r2 = 0.9322; F = 1374; df = 1, 100; P < 0.0001). The lower developmental threshold estimated from this equation was 11.23°C. Using this estimate of the lower developmental threshold, a thermal constant of 454.0 ± 8.1 degree days (DD) (n = 88) was calculated.
Figure 1. Effect of constant temperatures on developmental rate (d−1) of M. praeclarus when reared on E. kuehniella on detached tomato stems. Fitted curve: Lactin model (Lactin et al., Reference Lactin, Holliday, Johnson and Craigen1995).
Predatory capacity of M. praeclarus at different temperatures
Female M. praeclarus fed on significantly more E. kuehniella eggs than males (68% more, F = 18.453; df = 1, 153; P < 0.001) (fig. 2). For both sexes, the highest predation rate took place at 30°C followed by 25°C and 33°C, which was significantly higher than that obtained at 20°C (F = 26.909; df = 3, 153; P < 0.001). All adults died within 24 h of the experiment at 35°C, hence data of predation was not considered for this temperature. No interaction between sex and temperature was obtained (F = 1.583; df = 3, 153; P = 0.196).
Figure 2. Number (mean ± SE) of E. kuehniella eggs consumed by (a) females and (b) males of M. praeclarus at four constant temperatures and 14:10 h L:D. Bars topped by the same upper- and lower-case letters represent means that are not statistically different among temperatures and sexes, respectively (ANOVA P < 0.05). n = number of replicates.
Macrolophus praeclarus response to sweet pepper, eggplant and tomato odours
In the Y-tube olfactometer study, female M. praeclarus showed preference for the odours emitted from the three plants over the blank, no plant control treatment (χ2 = 4.829; P = 0.0280; χ2 = 8.758; P = 0.0031; χ2 = 5.121; P = 0.0236 for tomato, sweet pepper and eggplant, respectively) (fig. 3). When M. praeclarus females had a choice between tomato and sweet pepper, sweet pepper and eggplant or between tomato and eggplant, they showed no preference (χ2 = 1.882; P = 0.1701; χ2 = 1.400; P = 0.2367; χ2 = 1.324; P = 0.2498, respectively).
Figure 3. Response of M. praeclarus females in a Y-tube olfactometer to the odours of sweet pepper, eggplant and tomato. “n” indicates the number of individuals tested, “nc” indicates the number of individuals that did not make a choice and “n.s.” indicates non-significance. Significant differences based on a χ 2-test are marked with asterisk (P < 0.05).
Phytophagy by M. praeclarus
The number of N. tenuis that died inside the muslin bags during the phytophagy test was 3.8 ± 0.3 per replicate, which was significantly higher than that of 2.3 ± 0.5 of M. praeclarus (t 10 = 0.6948; P = 0.2515). Despite more individuals surviving, M. praeclarus did not produce necrotic rings, withered leaves, or other signs of visible damage on those tomato plants exposed to its feeding. In contrast, N. tenuis caused 3.2 ± 0.5 necrotic rings per replicate and 1.5 ± 0.4 withered leaflets on a fully expanded young leaf of the apical part of an entire tomato plant.
Plant defense response induction by M. praeclarus
Transcriptional analysis showed that the phytophagy of M. praeclarus on the apical part of tomato plants increased the expression of the PIN2 gene (JA pathway) (t 10 = 1.827; P = 0.0488), suggesting the activation of the related metabolic pathway (fig. 4b). In contrast, the ASR1 (ABA pathway) and the PR1 gene (SA pathway) expressions were not altered in M. praeclarus punctured plants compared to intact tomato plants (t 10 = 0.6948; P = 0.2515 and t 10 = 0.2063; P = 0.4202, respectively) (fig. 4a, c).
Figure 4. Expression of (a) ASR1, (b) PIN2 and (c) PR1 at the apical part of tomato plants either intact or exposed to the phytophagy of M. praeclarus. Transcription levels were normalized to the expression of EF1a measured in the same sample. Data are presented as the means ± SE of five biological replicates of transcription expression in relation to the housekeeping gene (n = 6). Asterisks on the bars show significant differences (P < 0.05) based on Tukey comparisons.
Discussion
To our knowledge, this is the first study detailing biological traits of the Neotropical zoophytophagous mirid, M. praeclarus. Macrolophus praeclarus was able to complete its development at four different temperatures (20, 25, 30 and 33°C) tested when supplemented with E. kuehniella eggs, but did not develop beyond the second nymphal instar under a constant temperature of 35°C. Our study indicates a low adaptability of M. praeclarus to extreme temperatures. This is known for tropical species, which are exposed to a narrow temperature range. These species are more affected by extreme temperature variations than species adapted to wider temperature regimes found in colder climates (Janzen, Reference Janzen1967; Rosner, Reference Rosner2013). Our results suggest that M. praeclarus may not perform well in warm climates. The results indicate M. praeclarus would likely be more successful as a biological control agent in temperate climates or in open field conditions, where it's optimal temperature range of 25–30°C occurs. However, our results were obtained under constant temperatures, which does not occur under field conditions where temperatures fluctuate throughout the day. In fact, M. praeclarus was able to establish and control B. tabaci effectively under the tomato growing conditions in South Florida that occur in spring and autumn (Roda et al., Reference Roda, Castillo, Allen, Urbaneja, Pérez-Hedo, Weihman and Stansly2020). In this study, the mean temperature was in the optimal range for M. praeclarus (25°–30°C), however, a maximum of more than 35°C and a minimum of less than 15°C had occurred.
The developmental time from egg to adult of M. praeclarus at 20°C (56.3 d) was considerably longer that reported for other species of Macrolophus. For example, M. caliginosus Wagner [syn. Macrolophus melanotoma (Costa)] took 47.7 days to develop on the whitefly Trialeurodes vaporariorum Westwood (Hemiptera: Aleyrodidae), M. pygmaeus took the same amount of time to develop on the aphid M. persicae and E. kuehniella, and M. basinicornis (Stal) needed 39.3 d to reach adulthood when reared on E. kuehniella eggs (Hart et al., Reference Hart, Tullett, Bale and Walters2002; Perdikis and Lykouressis, Reference Perdikis and Lykouressis2002; Martínez-García et al., Reference Martínez-García, Sáenz-Romo, Aragón-Sánchez, Román-Fernández, Sáenz-de-Cabezón, Marco-Mancebón and Pérez-Moreno2017; Bueno et al., Reference Bueno, Montes, Sampaio, Calixto and van Lenteren2018). The developmental parameters of mirids vary depending on the quality of the prey consumed (Sylla et al., Reference Sylla, Brévault, Diarra, Bearez and Desneux2016). The developmental time for M. praeclarus may decrease if more nutritious prey are available. As temperatures increase, the time required for M. praeclarus to complete the development from egg to adult was significantly reduced. At temperatures near 30°C, developmental times were comparable to other Macrolophus species studied. For example, Macrolophus praeclarus' developmental thermal constant of 454 DD is very similar to M. caliginosus (435 DD) (Hart et al., Reference Hart, Tullett, Bale and Walters2002), M. pygmaeus (455.1 DD) (Perdikis and Lykouressis, Reference Perdikis and Lykouressis2002) and M. basinicornis (476.2 DD) (Bueno et al., Reference Bueno, Montes, Sampaio, Calixto and van Lenteren2018). However, the lower temperature threshold for M. praeclarus (11.2°C) is higher than that of M. caliginosus (6.9°C), M. pygmaeus (7.2°C on T. vaporariorum or 8.7 on E. kuehniella) and M. basicornis (7.9°C).
We determined that M. praeclarus actively fed on E. kuehniella eggs. Given that E. kuehniella is a high-quality prey for predatory mirids, which is widely used in mass-rearing (Pérez-Hedo et al., Reference Pérez-Hedo, Riahi and Urbaneja2021), the predation rates found in this study may serve as a benchmark for establishing the mass-rearing conditions of M. praeclarus. Knowing the number of E. kuehniella eggs to be used can be not only interesting in mass rearing systems, but also when planning augmentative releases that may require supplementary food. The highest predation rates found in this study (40.7 eggs per female per day at 30°C) are similar to other mirid species when preying upon E. kuehniella (Mollá et al., Reference Mollá, Biondi, Alonso-Valiente and Urbaneja2014; Urbaneja-Bernat et al., Reference Urbaneja-Bernat, Mollá, Alonso, Bolkcmans, Urbaneja and Tena2015; Bueno et al., Reference Bueno, Montes, Sampaio, Calixto and van Lenteren2018). Egg predation, combined with recent studies showed that M. praeclarus controlled B. tabaci in tomato plants (Roda et al., Reference Roda, Castillo, Allen, Urbaneja, Pérez-Hedo, Weihman and Stansly2020), suggest the potential of this species as a biological agent. However, further studies are needed to determine whether M. praeclarus can control other key pests in horticultural crops, such as the lepidopteran T. absoluta and the thrips Frankliniella occidentalis Pergande (Thysanoptera: Thripidae). This point is important since it has been shown that the different species of the Macrolophus genus respond differently to crop pests. For example, M. basicornis is capable of reproducing successfully on T. absoluta eggs (Silva et al., Reference Silva, Bueno, Montes and van Lenteren2016; van Lenteren et al., Reference van Lenteren, Bueno, Burgio, Lanzoni, Montes, Silva and Hemerik2019) while M. pygmaeus does not (Mollá et al., Reference Mollá, Biondi, Alonso-Valiente and Urbaneja2014; Sylla et al., Reference Sylla, Brévault, Diarra, Bearez and Desneux2016).
One of the most desired traits when evaluating whether a mirid is a good candidate as a biological control agent is that its phytophagous behaviour does not result in damage to the crop (Pérez-Hedo et al., Reference Pérez-Hedo, Suay, Alonso, Ruocco, Giorgini, Poncet and Urbaneja2017; van Lenteren et al., Reference van Lenteren, Bueno, Burgio, Lanzoni, Montes, Silva and Hemerik2019; Pérez-Hedo et al., Reference Pérez-Hedo, Riahi and Urbaneja2021). Roda et al. (Reference Roda, Castillo, Allen, Urbaneja, Pérez-Hedo, Weihman and Stansly2020) showed that 30 nymphs per plant of M. praeclarus did not damage tomato plants in field cage experiments. Here, under controlled experimental conditions previously used to evaluate plant damage of other mirids species (Chinchilla-Ramírez et al., Reference Chinchilla-Ramírez, Pérez-Hedo, Pannebakker and Urbaneja2020), we showed that approximately eight M. praeclarus adults per leaflet caused no damage to tomatoes whereas approximately six adults of N. tenuis per leaflet did cause damage to the plant. These results are very encouraging since augmentative releases of M. praeclarus in tomato would likely not cause damage to the tomato plant. Thus, there would not be a need to continuously monitor populations or to control overpopulations as in the case of N. tenuis (Pérez-Hedo et al., Reference Pérez-Hedo, Riahi and Urbaneja2021).
Our results showed that M. praeclarus responds to tomato, sweet pepper and eggplant odours. An important aspect to determine their application as biological control agents would be to evaluate the survival rate of M. praeclarus has on each of these crops without the availability of prey and their capacity of these crops to retain mirid populations in periods of prey scarcity (Pérez-Hedo et al., Reference Pérez-Hedo, Riahi and Urbaneja2021). These applied aspects have direct implications for pest control as survival varies significantly between various species of mirids and crops (Perdikis and Lykouressis, Reference Perdikis and Lykouressis2002; Lucas and Alomar, Reference Lucas and Alomar2002a, Reference Lucas and Alomarb; Urbaneja et al., Reference Urbaneja, Tapia and Stansly2005; Abbas et al., Reference Abbas, Pérez-Hedo, Colazza and Urbaneja2014).
In this study, we showed that the phytophagy of M. praeclarus in tomato plants increases the expression of PIN2, a marker gene for jasmonic acid (JA) metabolic pathway. JA activation by other species of mirids, such as N. tenuis or M. pygmaeus, triggers the emission of HIPV's, in tomato and sweet pepper plants, which resulted in attraction to parasitoids (Pérez-Hedo et al., Reference Pérez-Hedo, Bouagga, Jaques, Flors and Urbaneja2015; Bouagga et al., Reference Bouagga, Urbaneja, Rambla, Flors, Granell, Jaques and Pérez-Hedo2018). The activation of this route can also increase the number of transcripts and protease inhibitors, which coincides with the reduced performance of various pests such as the whitefly B. tabaci on sweet pepper or two spotted spider mite on tomato Tetranychus urticae Koch (Acari: Tetranychidae) (Pappas et al., Reference Pappas, Steppuhn, Geuss, Topalidou, Zografou, Sabelis and Broufas2015; Bouagga et al., Reference Bouagga, Urbaneja, Rambla, Flors, Granell, Jaques and Pérez-Hedo2018; Pérez-Hedo et al., Reference Pérez-Hedo, Arias-Sanguino and Urbaneja2018). Furthermore, it has recently been confirmed that activation of this pathway by the phytophagy of N. tenuis also reduces tomato spotted wilt virus (TSWV) infection in sweet pepper (Bouagga et al., Reference Bouagga, Urbaneja, Depalo, Rubio and Pérez-Hedo2020). Further investigations of the effects that defensive activation mediated by M. praeclarus may have in tomato plants are warranted.
In summary, our results showed that M. praeclarus is well adapted to climatic conditions prevailing in the Neotropical region and provide valuable information for the development of the mass-rearing of this species. With the positive results obtained recently that show the capacity of M. praeclarus to establish on tomato plants where it effectively controls B. tabaci (Roda et al., Reference Roda, Castillo, Allen, Urbaneja, Pérez-Hedo, Weihman and Stansly2020), studies should focus on the development of tomato integrated pest management programmes for the Neotropical region of the American continent, based on the release of this zoophytophagous species.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0007485321000067.
Acknowledgements
This work was partly supported by the Spanish Ministry of Science and Innovation through the mobility stays abroad granted to MP-H (Jose Castillejo program - CAS18/00090) and to AU (Salvador Madariaga program - PRX18/00157) and through the project RTA2017-00073-00-00 and USDA PPA 7721 funding. The authors thank Dr Cory Penca (USDA APHIS PPQ) for helpful comments on earlier versions of the manuscript, Jean-Yves Berisse and Ana Redondo (SWFREC, UF) for their technical assistance, Dr Alejandro Tena (IVIA) for statistical advices and Ozgur Batuman (SWFREC, UF) for facilitating and allowing the conduct of some of the experiments in his laboratory. The findings and conclusions in this publication have not been formally disseminated by USDA and should not be construed to represent any Agency determination or policy. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. This work would not have been possible without the help and collaboration provided by the Phil Stansly family and especially his wife Silvia Stansly. Phil Stansly passed away in the course of preparing this work, so the authors would like to dedicate this work to him on a posthumous basis.
