Raoiella indica Hirst (Acari: Tenuipalpidae) is a polyphagous tenuipalpid mite that recently invaded the Western Hemisphere. It has a wide host-plant range mostly within the Arecaceae (palms), but it also attacks some plants within the Cannaceae, Pandanaceae, Musaceae, Heliconiaceae, Zingiberaceae, and Strelitziaceae (Carrillo et al. 2012a; Godim et al. 2013). Its major host is coconut, Cocos nucifera L., on which it reaches high infestation densities (ca. 4,000 individuals/leaflet [Peña et al. 2009]). Infestations found on other host plants are smaller but still problematic because the plants are important ornamental or native plants (Carrillo et al. 2012a; Godim et al. 2013).

The establishment of R. indica in the Caribbean has caused serious economic harm to coconut production with yield reductions estimated to be greater than 50% at some locations (CARDI 2010). Efforts have been made to identify natural enemies with potential in biological control programs targeting R. indica. Surveys for natural enemies conducted in the Philippines (Gallego et al. 2003), Tanzania and Benin (Zannou et al. 2010), India (Taylor et al. 2012), Mauritius (Hoy 2012), Reunion (Domingos et al. 2013), and also in islands of the Caribbean and Florida (Peña et al. 2009), Colombia (Carrillo et al. 2011), Venezuela (Vásquez & De Moraes 2012), Brazil (Domingos et al. 2013) and Mexico (Otero G. personal communication), indicate that R. indica has a different natural enemy complex at each site with only one predator species, Amblyseius largoensis (Muma) (Acari: Phytoseiidae), present in all the geographical areas (Carrillo et al. 2012b).

The predatory mite A. largoensis responded numerically to the arrival of R. indica in Florida (Peña et al. 2009), which motivated detailed studies on the efficiency of this predator. An initial study determined that this predator was able to feed, develop, and reproduce on a diet consisting solely of R. indica showing ‘better’ reproductive parameters than when fed other prey and pollen (Carrillo et al. 2010). Follow-up studies determined that A. largoensis responded both functionally and numerically to increasing densities of R. indica, and showed a marked preference for eggs over larvae, nymphs, and adults of the phytophagous mite (Carrillo & Peña 2012). Further studies examined the response of 4 populations of A. largoensis with previous exposure to R. indica or Tetranychus gloveri Banks (Acari: Tetranychidae) (Carrillo et al. 2012c). The 4 populations of A. largoensis, including predators never exposed to R. indica, were likely to accept and consume high numbers of R. indica eggs. However, predators previously exposed to R. indica were more likely to feed on R. indica larvae than were naïve predators, suggesting that Amblyseius largoensis may be responding to the invasion by R. indica either by learning or evolving genetically to be better predators of this pest (Carrillo et al. 2012c). The combined previous studies indicate that A. largoensis is actively responding to the invasion by R. indica and provide a framework to hypothesize that this predator has the potential to be used in biological control and IPM programs targeting R. indica. The objective of this study was to provide a quantitative evaluation of the ability of A. largoensis to reduce R. indica densities at 4 predator-prey ratios.

Materials and Methods

The experimental approach combined exclusion and release techniques to obtain different levels of A. largoensis on coconut palms infested with R. indica. The experiment was conducted inside a climate-controlled 43.2 m² glasshouse (26.5 ± 4 °C, RH 70 ± 20%) located at the Tropical Research and Education Center in Homestead, Florida (N 25° 35.49′ W 80° 04.03′). Thirty-two 1-year-old potted, pest-free and unsprayed Malayan dwarf coconut palms (about 1.5 m tall) were used in this experiment. A middle frond in each palm was selected as the experimental unit and remaining fronds were manually removed. A yellow “tagging” tape (∼5 cm wide) coated with Tanglefoot® was tied around the base of the frond to exclude any crawling arthropods. Potted palms were arranged on benches and clear plastic sheets (1.5 × 3 m) were hung from the roof of the glasshouse to isolate each individual palm. Fronds were then inspected with a hand lens (20×) and any unwanted arthropods (i.e., Aonidiella orientalis Newstead [Hemiptera: Diaspididae], Tetranychus gloveri Banks [Acari: Tetranychidae], A. largoensis) were removed by hand every 3 days during 2 consecutive weeks before being inoculated with R. indica. Raoiella indica specimens used for infestation were obtained from a greenhouse colony of the red palm mite (reared as per Carrillo et al. 2010). Two R. indica-infested pinnae (containing ca. 30 mites) were then attached to the abaxial surface of each palm frond with a hair clip. Infested palms were left undisturbed for 30 days. After this period, R. indica establishment was evaluated by counting the number of motile stages and eggs per pinna using a hand lens (20 ×).

Treatments were allocated to each palm depending on the degree of infestation. Four A. largoensis release rates (0 = control, 1:10, 1:20 and 1:30 A. largoensis: R. indica) were tested with 8 replicates per treatment. Predators were obtained from a coconut palm plantation (Malayan dwarf variety, pesticide-free, N 26° 02.93′ W 80° 09.82′) that had been infested with R. indica since 2007, so the predators were experienced on this prey. Predators were confirmed as A. largoensis and maintained on rearing arenas containing R. indica under laboratory conditions for 1 week before testing. Predators were then transferred from the rearing arenas to the experimental coconut fronds using a camel hair brush. After this, all fronds were carefully inspected every 2 weeks with a 20 × hand lens and any undesirable predators or phytophagous arthropods removed. Three months later, fronds were excised, taken to the laboratory, and the total number of R. indica and A. largoensis eggs and motiles per pinna were inspected under a dissecting microscope (50 ×). The surface area of each of the pinnae was estimated by measuring its width and height and calculating the area using ½ base × height in order to account for the triangular shape of the pinnae. Leaf damage was quantified by measuring the width and height of necrotic areas and calculated in the same manner.

Results

One month after inoculation, and before predators were released, R. indica mean infestation levels ranged from 41.3 ± 14.6 to 46.6 ± 16.5 (mean ± SEM) R. indica motile stages per pinna (Table 1). Random allocation of treatments ensured similar infestation levels between treatments before predators were released (F = 0.15; df = 3, 8; P = 0.92). The number of predators released ranged from 0 (control treatment) to 4.1 ± 1.1 A. largoensis females per pinna according to the release rates and the infestation level (Table 1). Three months after predators were released, R. indica mean infestation levels ranged from (124.7 ± 94.1 to 999.6 ± 94.6) R. indica motile stages per pinna (Table 1).

The release of A. largoensis resulted in a significant reduction in R. indica densities relative to the densities found on predator-free palms (df = 3, 28; F = 7.76; P < 0.001). Raoiella indica reached densities of 70.7 ± 7.2 individuals/cm² (mean ± SEM) in predator-free palms, and 5.3 ± 7.1, 31.7 ± 5.7 and 40.4 ± 5.9 (individuals/cm²) when A. largoensis was released at the rates of 1:10, 1:20 and 1:30 A. largoensis: R. indica, resulting in a 92, 54 and 42 percent reduction of R. indica densities, respectively, by the end of 3 months (Fig. 1).

Table 1.

Levels of infestation of Raoiella indica (Mean ± sem) 1 month after being established on predator- free coconut palms and just before Amblyseius largoensis predators were introduced at various predator to prey ratios; and the population levels of prey and predators three months later. the experiment was conducted at Homestead, Florida in a climate-controlled glasshouse (26.5 ± 4 °c, rh 70 ± 20%).

Raoiella indica egg densities on predator-free palms (19.3 ± 2.2 individuals/cm², mean ± SEM) were similar to those found on palms where predators were released at 1:30 A. largoensis: R. indica (13.9 ± 1.8 individuals/cm²); however, these were significantly greater than the egg densities found on palms where predators were released at 1:10 and 1:20 (3.3 ± 2.2 and 11.6 ± 1.7). Predator releases resulted in an 82, 39 and 27 percent reductions in R. indica egg densities in the 3 A. largoensis/R. indica release rates, respectively (df = 3, 28; F = 5.13; P < 0.01) (Fig 1A). More motile stages of R. indica were present than eggs on the treatments with the 2 lowest A. largoensis release rates and on the predator-free palms. In contrast, R. indica populations consisted of more R. indica eggs than motile stages in the treatment with the highest A. largoensis release rate (Fig 1C), suggesting that the predators fed preferentially on eggs. Motilestage densities in predator-free palms (51.5 ± 5.9 R. indica motiles/cm², mean ± SEM) were significantly higher than those found in the 3 release treatments (2.0 ± 5.8, 20.1 ± 4.7 and 26.5 ± 4.9 R. indica motiles/cm², mean ± SEM)), resulting in 96, 61 and 48 percent reduction of R. indica motile stages in the 1:10, 1:20 and 1:30 A. largoensis: R. indica release rates, respectively (df = 3, 28; F = 6.87; P < 0.001) (Fig 1B).

Amblyseius largoensis established on all palms where it was released and was not detected in the control treatments, indicating that the isolation methods used were successful over the 4 months of the experiment. However, the numbers of A. largoensis recovered in the final destructive sampling (0.25 ± 0.8, 0.36 ± 1.4, 0.4 ± 1.3 and 0 ± 0 predators per/pinna, in the 1:10, 1:20 and 1:30 A. largoensis: R. indica release rates, respectively) were lower than the numbers released (Table 1), and did not vary along the release treatments (df = 3, 28; F = 10.04; P < 0.001). Besides A. largoensis, no other predators were detected.

The leaf area per pinna and the number of pinnae per palm were equivalent among the palms assigned to each A. largoensis release rate (F = 0.69 P = 0.56 and F = 0.11.4 P =0.95, respectively). In contrast, the percentage of leaf area showing necrosis was significantly higher in palms where A. largoensis was excluded compared to palms where the predator was released (χ² [3, N = 32] = 99.83, P < 0.0001).

Fig 1.

Effect of 4 release rates of A. largoensis (0= control, 1:10, 1:20, 1:30 A. largoensis/R. indica) on R. indica densities. A. R. indica egg density B. R. indica motile stages density, C. R. indica total density showing the proportion of eggs and motile stages. Error bars represent standard errors.

Discussion

Convincing evidence of the efficacy of a natural enemy requires not only quantitative evaluations of pest densities with and without the presence of the natural enemy, but also clear separation of the mortality caused by the natural enemy from that of other biotic and abiotic factors (Luck 1988; Van Driesche & Bellows 1996). In our experiment, exclusion tactics (sticky barriers, hand removal and cages) were used to quantify R. indica densities in palms with different ratios of A. largoensis, including predator-free palms. No other predators were detected and since palms were kept under controlled environmental conditions, the observed differences in R. indica densities could be attributed to A. largoensis alone.

Previous studies indicated that A. largoensis has a marked preference for R. indica eggs and has difficulty feeding on the motile stages (Carrillo & Peña 2012). In this experiment, pest density reductions were observed both in the egg and motile stages of R. indica. However, the proportion of eggs and motile stages in the treatment with the highest release rate, where the greatest pest reduction was observed, differed significantly from all other treatments. This result could have been caused by a higher predation of eggs in this treatment that resulted in fewer individuals developing into motile stages. By contrast, the treatments with the 2 lowest release rates and the predator-free treatment resulted in an accumulation of motile stages and substantially higher prey densities. It is possible that at low predator densities more R. indica individuals completed the egg stage and became larvae, a stage when they are less likely to be preyed upon by A. largoensis. These results suggest that A. largoensis can be effective in controlling R. indica only when it consumes a large proportion of R. indica eggs, and consequently reduces the accumulation of motile stages. Moreover, the ability of A. largoensis to suppress R. indica might depend not only on the predator - prey ratio but also on the population density of R. indica. It is possible that, as for other generalist predators (i.e., as in James 1990; McMurtry 1992), A. largoensis could have its major effect at low population densities of R. indica.

Predation on R. indica by A. largoensis resulted in a reduction of the coconut leaf area showing damage. The proportion of damaged leaf tissue differed significantly between treated and untreated palms, but not among the 3 release rates tested. Predator-free palms showed necrosis 4 months after being inoculated with R. indica. Therefore, the experiment was concluded at that time in order to prevent advanced necrosis and subsequent reduction of host-plant quality, which would eventually affect R. indica survivorship.

Exclusion tactics were useful to quantify R. indica densities in palms with different levels of A. largoensis. However, a relatively low number of predators were recorded after the fronds were destructively sampled, suggesting that the physical barrier could have affected the establishment and reproduction of the predators. An alternative to exclusion by the physical means used in our experiment is the use of acaricides to reduce mite numbers (Braun et al. 1987; McMurtry 1992; Cuthbertson et al. 2003). Some chemicals are highly toxic to predators but less toxic to phytophagous mites (Roush & Hoy 1978), so they can be used for predator exclusion purposes. However, the use of pesticides has often led to problems in interpreting results because of possible pesticide-induced physiological effects on the plants (Jones et al. 1983), pesticide-induced sex ratio bias and stimulation of the reproductive potential of the prey population (hormoligosis) (Bartlett 1968; Dittrich et al. 1974; Hoy et al. 1979; Maggi & Leigh 1983; Cuthbertson et al. 2003). Despite these problems, acaricidal disruption is a quick and easy way to evaluate the effect of natural enemies on a large scale (Luck 1988). Thus, the feasibility of using this technique to test the effects of different of A. largoensis release rates on R. indica populations under natural conditions should be investigated.

Raoiella indica populations in Florida have shown a steady decline that could be related to the subtropical conditions of the state as well as the build-up of predators, primarily A. largoensis (Duncan et al. 2010). However, R. indica populations in the Caribbean and in areas of recent introduction are still causing damage, which suggests that additional mortality factors may be needed to effectively suppress the populations of this invasive pest, especially mortality factors affecting motile stages of R. indica. Assis et al. (2013) identified acaricides that are selective for A. largoensis such as fenpyroximate and spirodiclofen, which could be used to integrate chemical and biological control strategies against R. indica. In addition, provision of alternative food sources could increase predator populations resulting in a better potential control of R. indica.

In conclusion, our results suggest that A. largoensis is an important mortality factor that can reduce R. indica densities and the damage inflicted on coconut fronds under greenhouse conditions. Moreover, the search for additional natural enemies, including fungal pathogens should be intensified to suppress the populations of this invasive pest in tropical areas, and areas recently invaded by this pest.