Evaluation of potential natural enemies of hibiscus mealybug, Nipaecoccus viridis (Hemiptera: Pseudococcidae) in Florida citrus

1 Introduction

Hibiscus mealybug (Nipaecoccus viridis Newstead; Hemiptera: Pseudococcidae), also known as lebbeck mealybug or spherical mealybug, is an emerging and serious pest in Florida citrus. Originally discovered in Florida on dodder (Cuscuta sp.; Convolvulaceae) in 2009 (Stocks and Hodges 2010), hibiscus mealybug was first identified on citrus in 2019 (Diepenbrock and Ahmed 2020) and has since spread to many citrus groves in central and southern regions of the state. Infestations of hibiscus mealybug can cause fruit distortions, fruit drop, branch dieback, and can even kill young trees if infestations are severe (Cilliers and Bedford 1978; Diepenbrock and Ahmed 2020; Franco et al. 2004).

While hibiscus mealybug numbers can reach heavily damaging levels, a suite of natural enemies usually keeps this pest in check in its native range (Cilliers and Bedford 1978; Diepenbrock and Ahmed 2020; Franco et al. 2004; Sharaf and Meyerdirk 1987). Numerous predators have been found to prey on hibiscus mealybug. A total of 21 species of coccinellids (Coleoptera) including Cryptolaemus montrouzieri (Mulsant) (Nechols and Seibert 1985; Kairo et al. 2013), Nephus arcuatus (Kapur) (Zarghami et al. 2016), and Scymnus roepki (Fluiter) (Nechols 2003), have been reported to feed on the hibiscus mealybug (García Morales et al. 2016). Other predators recorded in the literature include neuropteran larvae such as Sympherobius sp. (Hemerobiidae; Cilliers and Bedford 1978) or larvae belonging to the Chrysopidae family (Neuroptera; three species), and various dipteran larvae belonging to the families Drosophilidae (four species), Cecidomyiidae (five species), and Chamaemyiidae (one species) (Cilliers and Bedford 1978; García Morales et al. 2016; Nechols and Seibert 1985; Nechols 2003; Stocks 2013). Parasitoids from the family Encyrtidae totaling more than 55 species including Anagyrus indicus (syn. Anagyrus agraensis) Shafee, Alam & Argawal (Hymenoptera: Encyrtidae) can also play an important role in regulating hibiscus mealybug populations (García Morales et al. 2016; Meyerdirk et al. 1988; Nechols and Seibert 1985; Nechols 2003). Predators and parasitoids such as these often preclude the need for more intensive management.

Unfortunately, management practices that disrupt natural enemy communities can lead to damaging outbreaks of hibiscus mealybug. Applications of broad-spectrum insecticides to control hibiscus mealybug in citrus have historically been ineffective and have instead worsened outbreaks by harming natural enemy communities (Franco et al. 2004; Sharaf and Meyerdirk 1987). Mealybug outbreaks that occurred in South African citrus production were linked to applications of insecticides that killed off natural enemies (Cilliers and Bedford 1978), and outbreaks were controlled only when mealybug-specific natural enemies re-established after insecticide applications ceased (Franco et al. 2004). Using protective screens or mesh to exclude pest insects may also worsen infestations of hibiscus mealybug (Diepenbrock and Ahmed 2020). Protective mesh bags placed over individual citrus trees led to increased scale infestations (Gaire et al. 2022), and hibiscus mealybug infestations found inside of exclusion screens often appear worse compared to those outside of the protective screening (Middleton & Diepenbrock personal observation). Exclusion bags placed on pomegranates in India led to increased hibiscus mealybug infestations compared to unbagged fruits (Shevale 1994). Taken together, chemical control and protective screens/mesh may worsen outbreaks by excluding natural enemies.

In Florida citrus, over a decade of chemical management to control Asian citrus psyllid (Diaphorina citri Kuwayama; Hemiptera: Liviidae) has likely reduced the abundance of natural enemies. Insecticidal sprays used to manage Asian citrus psyllid led to significant reductions in spider, arboreal ant, and coccinellid populations (Monzo et al. 2014; Qureshi and Stansly 2007). Broad-spectrum insecticides such as chlorpyrifos and foliar applications of neonicotinoids such as imidacloprid are commonly used in citrus production in Florida (Diepenbrock et al. 2021) and have been shown to be toxic to natural enemies including coccinellids, lacewings, and parasitoids (El-Mageed et al. 2018; Hall and Nguyen 2010; Qureshi and Stansly 2007). Considering many of the predators in Florida citrus are coccinellids and lacewings (Michaud 2002), current management practices are likely a factor in reducing the abundance of predators and allowing hibiscus mealybug to become established as a pest in Florida in recent years. Additionally, many Florida growers have taken to covering young trees with individual protective covers (IPCs) (Alferez et al. 2021) and growing citrus in screenhouses (citrus under protective screening or CUPS) (Schumann et al. 2021) to exclude Asian citrus psyllid and the huanglongbing disease it vectors. This process of bagging trees and producing citrus under protective screening both excludes many natural enemies and may worsen mealybug infestations.

To achieve sustainable management of hibiscus mealybug in Florida, it is necessary to determine which naturally occurring predators consume hibiscus mealybug. Additionally, commercially available predators that could be purchased for augmentative releases need to be evaluated. This information would allow natural enemies to take hold and gain control of mealybug outbreaks without resorting to chemical control options that often are insufficient (Diepenbrock, personal observation). Additionally, identifying which naturally occurring predators in Florida citrus prey on hibiscus mealybug can incentivize growers to conserve existing natural enemy populations and help inform better conservation biological control practices. Predators that can consume hibiscus mealybug ovisacs are of particular interest as the ovisac wax can protect the eggs inside from contact insecticides (Sharaf and Meyerdirk 1987), making chemical control difficult. Finally, field-testing releases of promising predators to determine if they reduce mealybug infestations within groves is necessary to determine if augmentative biological control is a viable option.

We evaluated five commercially available predators and six wild-caught predators to determine if they would consume different life stages of hibiscus mealybug. Additionally, we collected and dissected mealybug clusters from infested groves to assess which predators and parasitoids are actively preying on hibiscus mealybug in Florida citrus. Finally, we evaluated an augmentative release of Cr. montrouzieri inside of a commercial CUPS production system to determine how this would impact infestations of hibiscus mealybug.

Our study addressed the following questions:

1. Which commercially available predators will feed on hibiscus mealybug nymphs and ovisacs under laboratory conditions?

2. Which naturally occurring predators in Florida will feed on hibiscus mealybug nymphs and ovisacs under laboratory conditions?

3. Which natural enemies are found actively preying on hibiscus mealybug in Florida groves?

4. Can augmentative releases of Cr. montrouzieri affect populations of hibiscus mealybug in a commercial CUPS production system?

2 Materials and methods

2.2 No choice assays

Hibiscus mealybugs were reared in a laboratory colony on Volkamer lemon trees and kept at approximately 27 °C. Two different life stages of hibiscus mealybug were used in predator assays: 2nd–3rd instar nymphs, and adult females with ovisacs. Arenas for mealybug nymphs were prepared by excising Volkamer lemon leaves and placing the petiole in a 1.5 ml microcentrifuge tube filled with deionized water and wrapped in plastic wrap (Parafilm^®, Bemis Company, Inc., Sheboygan Falls, Wisconsin). Leaves were then placed into individual 150 × 15 mm polystyrene Petri dishes (Thermo Fisher Scientific, Hampton, New Hampshire), and 2nd–3rd instar mealybugs were added to the leaves using a fine tipped brush (size 2 camel hair, Torrington Brush Works, Connecticut). Arenas for ovisacs were prepared by placing a single adult female or late 3rd instar nymph on the excised leaf and allowing it to develop an ovisac over the course of 1–3 weeks.

For the duration of all tests, predators were kept at 28 ± 2 °C, 16:8 h L:D and 60 ± 20 % relative humidity. For commercially purchased predators, a subset (n = 5) of the population was placed in arenas with known quantities of frozen flour moth, Ephestia kuehniella (Zeller; Lepidoptera: Pyralidae) eggs, and the number of eggs consumed was recorded each day for 3 days. Ep. kuehniella eggs, both fresh and frozen, are commonly used for the maintenance of colonies of generalist arthropod predators. These eggs were used as a positive control to ensure predators from the same cohort would consume prey. For wild-caught predators, individuals were given Ep. kuehniella eggs when first brought to the laboratory and observed to ensure they were actively consuming prey. This was performed as predators were collected from actively managed citrus groves where exposure to pesticides could impede their consumptive ability. Individual predators were then starved for 24 h prior to testing.

To determine how many 2nd and 3rd instar nymphs should be given to each predator, 5–20 nymphs were presented to a subset of each predator species (n = 3), and the number of mealybugs consumed over the course of a day was recorded. Based on these initial results, five nymphs were used for Or. insidiosus adults, 10 nymphs for Hi. convergens, Di. austrinus, Co. septempunctata, and Ad. bipunctata adults, and 20 nymphs for Cr. montrouzieri adults and larvae, Cu. coeruleus adults, Ceraeochrysa sp. larvae, Ch. carnea larvae, and Eu. annulipes adults. Cr. montrouzieri adults frequently consumed all 20 mealybugs provided. To determine how many mealybugs Cr. montrouzieri would consume if provided mealybugs ad libitum, a subset (n = 5) was given 40–60 2nd and 3rd instar nymphs. As a negative control, 20 nymphs were placed into an arena without a predator.

For trials with 2nd and 3rd instar nymphs, starved predators were introduced to arenas with mealybug nymphs along with a water-soaked cotton ball. The number of living, dead, and absent mealybugs were assessed daily for 3 days. Mealybugs that were absent were considered to have been consumed. In the case of piercing/sucking predators, mealybugs that were drained of fluids also were classified as consumed. All mealybug nymphs that were not consumed were assessed for mortality by gently probing with a fine tipped brush. Mealybugs that moved in response were considered alive, and those that did not were classified as dead. Mealybugs that had been clearly attacked (i.e. showing injuries such as parts missing or sides ripped off or having fluids coming out of their body) and killed by predators, but were not consumed, also were counted as dead. All dead and consumed mealybugs were replaced daily to keep the number of available living mealybugs constant.

For trials with ovisacs, all predators were placed in arenas with a water-soaked cotton ball and a single ovisac. Mealybugs were checked daily for 3 days. Ovisacs were considered consumed if over half of both the adult and ovisac were eaten, or if the adult and ovisac were drained of fluids. For adults and ovisacs that were not consumed, adult mealybugs were gently probed each day with a fine tipped brush to assess mortality. Mealybugs that moved in response were considered alive, and those that did not were classified as dead. Additionally, predator mortality was assessed every day by observing for movement, and by probing with a fine tipped brush if necessary. Predators that remained immobile were considered dead. The number of replicates (N) set up for each predator varied and can be found in Tables 1 and 2.

Table 1:

Hibiscus mealybug nymphs consumed or dead (average number ± standard error), and the percentage of adult with ovisacs that were consumed or died, separated by predator species. A treatment with no predator in the arena also was tested and served as negative control.

Predator		Nymphs			Adult with ovisacs
		N	Consumed	Dead	N	% consumed	% dead
Commercially available
	Cryptolaemus montrouzieri adults	18	18.74 ± 0.19 a	0.19 ± 0.07 f	21	100 a	0 c
	Cryptolaemus montrouzieri larvae	13	13.33 ± 0.71 c	1.54 ± 0.29 bc	21	90.5 ab	4.8 c
	Chrysoperla carnea larvae	20	16.50 ± 0.86 b	1.55 ± 0.39 bcde	20	30 cd	55 a
	Adalia bipunctata adults	20	4.6 ± 0.45 e	1.98 ± 0.25 b	16	0 e	12.5 bc
	Hippodamia convergens adults	21	1.63 ± 0.21 f	4.17 ± 0.26 a	21	0 e	13.6 bc
	Orius insidiosus adults	14	0.31 ± 0.12 g	0.60 ± 0.09 e	10	0 de	0 bc
Naturally occurring
	Euborellia annulipes adults	20	14.60 ± 1.08 bc	0.65 ± 0.19 ef	23	73.9 b	0 c
	Ceraeochrysa sp. larvae	18	14.07 ± 0.99 bc	0.84 ± 0.26 de	24	66.7 bc	12.5 bc
	Curinus coeruleus adults	20	12.5 ± 0.88 c	2.48 ± 0.36 b	21	0 e	9.5 bc
	Harmonia axyridis adults	18	8.41 ± 1.08 d	4.48 ± 0.53 a	16	0 e	31.3 ab
	Diomus austrinus adults	11	3.94 ± 0.82 e	1.61 ± 0.40 bcd	10	10 de	0 bc
	Coccinella septempunctata adults	17	3.49 ± 0.41 e	4.08 ± 0.26 a	18	11.1 de	55.6 a
Control		20	0.2 ± 0.06 g	0.98 ± 0.15 cd	20	0 e	0 c

1. *Different letters denote significant differences at a level of p < 0.05 (pairwise Wilcox tests) between values in the same column. N = number of predators tested (replicate).

Table 2:

Percentage of predators that died in tests with hibiscus mealybug nymphs and ovisacs.

Predator type		Scientific name		Nymphs		Ovisacs
				N	% dead	N	% dead
Commercially Available			Cryptolaemus montrouzieri	20	10 b	22	4.55 bc
			Cryptolaemus montrouzieri larvae	13	0 b	21	0 c
			Chrysoperla carnea	20	5 b	22	9.09 bc
			Adalia bipunctata	20	0 b	23	30.43 ab
			Hippodamia convergens	26	19.23 b	24	8.33 bc
			Orius insidiosus	33	75.76 a	23	56.52 a
Naturally Occurring			Euborellia annulipes	20	0 b	23	0 c
			Ceraeochrysa sp. larvae	18	0 b	26	7.69 bc
			Curinus coeruleus	21	4.76 b	21	0 c
			Harmonia axyridis	23	26.09 b	20	20 abc
			Diomus austrinus	11	9.09 b	12	16.67 ab
			Coccinella septempunctata	20	20 b	21	14.29 bc

1. *Different letters denote significant differences at a level of p < 0.05 (pairwise Wilcox tests) between values in the same column. N = number of predators tested (replicate).

For all trials with 2nd and 3rd instar nymphs, we compared the average number of nymphs consumed per day across all predator species. Additionally, to determine mealybug mortality, we compared the average number of dead mealybugs that were not consumed per day across all predator species. Commercially available and naturally occurring predators were analyzed together. The number of mealybugs consumed by each predator and the number of dead mealybugs did not meet the assumptions of homogeneous variance or normality and were compared using Kruskal Wallis tests. Pairwise Wilcox tests with a Benjamini & Hochberg adjustment were used to determine means separation between individual predator species. Predator mortality was similarly assessed with Kruskal Wallis tests, and pairwise Wilcox tests with a Benjamini & Hochberg adjustment were used to determine means separation between individual predator species.

For all trials with ovisacs, the proportion of predators that consumed ovisacs and the proportion of adult female mealybugs that died but were not consumed were calculated and compared using Kruskal Wallis tests. Pairwise chi-squared tests were used to compare between individual species. Predator mortality was similarly assessed with Kruskal Wallis tests, and pairwise Wilcox tests with a Benjamini & Hochberg adjustment were used to determine means separation between individual predator species. All tests were carried out in R (R Core Team 2022).

3 Results

3.3 Field release of Cr. montrouzieri

A significant reduction in hibiscus mealybug infestations was detected (χ ² = 105.73, df = 2, p < 0.0001) 16 days and 37 days after release of Cr. montrouzieri (Figure 1). Of approximatively 2,300 trees inspected on 3 August 2021, 99 were either severely (score = 2) or lightly (score = 1) infested. Of those 99 trees, 55 were still infested on 20 August 2021, and 34 were still infested on 10 September 2021 with infestations ranging between light and severe. Distribution of mealybugs was patchy throughout the CUPS production system for the duration of this study. Significantly more Cr. montrouzieri larvae were found 16 days after release compared to other sampling dates (χ ² = 102.23, df = 2, p < 0.0001), with 57.7 % of infested trees having at least one Cr. montrouzieri larva on 20 August 2021 compared to 12.4 % on 10 September 2021, and 0 % on 3 August 2021. The percentage of infestations that appeared undamaged was significantly lower after the release of Cr. montrouzieri (χ ² = 219.21, df = 2, p < 0.0001), with 100 % undamaged infestations on 3 August 2021 compared to 13.4 % on 20 August 2021 and 8.6 % on 10 September 2021.

Figure 1:

Short-term effect of augmentative release of Cryptolaemus montrouzieri on hibiscus mealybug infestations (average ± S.E) on trees identified as infested on 3 August 2021 in a commercial CUPS production system 0, 17, and 38 days after release of predators. Different letters denote significant differences at a level of p < 0.05 (pairwise Wilcox tests).

When looking at a longer timescale and scouting the entire grove for infestations, we observed a significant decrease in hibiscus mealybug infestations at 63 (z = −3.80, p = 0.00015) and 106 days (z = −2.56, p = 0.01035) after release of Cr. montrouzieri, although there was no significant difference between infestations 63 and 106 days after release (z = 1.3, p = 0.19387) (Figure 2). Sixty trees were infested on 5 October 2021, and 76 trees were infested on 17 November 2021. There was no effect of date on the number of Cr. montrouzieri larvae (χ ² = 2, df = 2, p = 0.3679) with only a single larva found across all three sampling dates. There was a significant effect of date on the number of undamaged infestations (χ ² = 37.087, df = 2, p < 0.0001), with 55 % undamaged infestations on 5 October 2021 and 84.2 % on 17 November 2021.

Figure 2:

Long-term effect of augmentative release of Cryptolaemus montrouzieri on hibiscus mealybug infestations across all trees (average ± S.E) in a commercial CUPS production system 0, 63, and 106 days after release. Different letters denote significant differences at a level of p < 0.05 (generalized mixed effects models).

4 Discussion

Of the commercially purchased predators, only Cr. montrouzieri consistently consumed both hibiscus mealybug nymphs and ovisacs in no-choice trials. Additionally, a reduction in mealybug populations inside a CUPS production system was observed after the release of Cr. montrouzieri. Nevertheless, because we did not include a control plot (i.e., trees where Cr. montrouzieri was not released) in the field release experiment, we cannot directly correlate the decrease in mealybug infestation with the presence of Cr. montrouzieri. Indeed, the reduction in infestation could be due to other factors such as the presence of other natural enemies or abiotic factors (e.g. temperature, humidity) that negatively impacted mealybug populations. The efficacy of Cr. montrouzieri on several mealybug species including hibiscus mealybug has been documented (Kairo et al. 2013; Khan et al. 2012; Qin et al. 2019), and Cr. montrouzieri has been used as an effective biological control agent of numerous other mealybug species (Kairo et al. 2000, 2013). Our results showed evidence that the decrease in hibiscus mealybug numbers corresponds with the release of Cr. montrouzieri.

Moreover, our experiment was conducted on an ad hoc basis in conjunction with the grower who owned the screen house, and our methods did not fully capture the effect that Cr. montrouzieri had on mealybug infestations. In almost all instances where infestations appeared undamaged after beetles were released consisted of single ovisac infestations, suggesting that the beetles may be more attracted to large clusters than isolated ovisacs. Larger infestations of ovisacs produce a greater quantity of honeydew, which is a known attractant for coccinellids, so this finding is not surprising (reviewed in Lundgren 2009). Additionally, our system of grading infestations did not account for instances where infestations graded as a “1” were almost entirely consumed, but at least one ovisac remained, and therefore were still graded as a “1”. This resulted in a more conservative measure of Cr. montrouzieri efficacy and helps explain what appears to be a resurgence in mealybug infestations 3 months after predator release. While the number of hibiscus mealybug infestations slightly increased by 17 November 2021, it remained statistically lower than on 3 August 2021, before releasing Cr. montrouzieri. In addition, almost all new infestations consisted of single ovisacs, whereas on 3 August 2021, most infestations graded as a “1” consisted of dozens of ovisacs at least, spread around the tree. Further studies including a control plot and a method with more comprehensive ways of quantifying mealybug infestations are necessary to confirm that Cr. montrouzieri can control hibiscus populations under field conditions.

Among the other commercially available predators, Ch. carnea also consumed hibiscus mealybug nymphs at high rates, but only infrequently consumed ovisacs. As a highly generalist predator, it follows that Ch. carnea would feed on hibiscus mealybug nymphs and has previously been documented consuming Phenacoccus solenopsis (Tinsley) nymphs at comparable rates to what we found with Ni. viridis (Khan et al. 2012; Rashid et al. 2012). However, we anticipated Ch. carnea would similarly consume adults and ovisacs, especially because their piercing-sucking mouthparts should be able to bypass the protective wax on adults and ovisacs. For almost all Ch. carnea tested, we either directly observed them attacking the ovisac, and/or puncture wounds were found in the adult mealybugs. Adult mealybug mortality was high in tests with Ch. carnea, further confirming our observations. Despite recognizing adults and ovisacs as prey and successfully attacking them, it appears that ovisacs and adults are unpalatable to Ch. carnea in ways that the nymphs are not. The dense wax present on adults and ovisacs compared to early instar nymphs that lacked coating likely acts as a physical barrier, reducing the effectiveness of the predators but also hindering their feeding behavior (Barners 1975; Gonçalves-Gervásio and Santa-Cecília 2001). Several studies reported better performance of lacewings on first instar mealybug compared to adult mealybug due to this phenomenon (Hameed et al. 2013; Khan et al. 2012; Rashid et al. 2012). It is possible their protective wax was a greater deterrent than expected, or that the adult mealybugs themselves contain unpalatable compounds. For example, Hodek (1996) suggested that Rodolia cardinalis (Mulsant) (Coleoptera: Coccoidea) rejected Icerya purchasi Maskell (Hemiptera: Monophlebidae) both on the host plants and isolated from the plants because the plant substances ingested by the prey (e. g. the alkaloid, sparteine) made I. purchasi unpalatable for the ladybeetle. Mealybugs also can secrete some defensive fluids through their ostioles (Williams 1978). For instance, Gillani and Copeland (1999) showed that Pseudococcus longispinus (Targioni Tozzetti) (Hemiptera: Pseudococcidae) secretes droplets when attacked by the brown lacewing, Sympherobius fallax Navas (Neuroptera: Hemerobiidae). The secretion solidifies and clogs the predator mouthparts leading to its death by starvation.

Other commercially purchased predators consumed very few hibiscus mealybug nymphs, and consumed no ovisacs. Generalist predators with chewing mouthparts like Hi. convergens and Ad. bipunctata may have been deterred by the sticky wax covering adult mealybugs and ovisacs. Additionally, while both Hi. convergens and Or. insidiosus were found preying on hawthorn mealybug nymphs in a previous study, they did not consume adult mealybugs (Cranshaw et al. 2000). This may be because small predators like Or. insidiosus would have difficulty consuming entire ovisacs or even adult hibiscus mealybugs. It was more unexpected that none of these predators fed on hibiscus mealybug nymphs to any meaningful degree. In tests with Hi. convergens, mealybug nymph mortality was significantly higher than controls, with many of the dead mealybugs ripped apart, or showing clear signs of having been chewed. This indicates that Hi. convergens recognized the nymphs as potential prey, but often found them unpalatable. Like Ch. carnea, which attacked but did not frequently consume adult mealybugs and ovisacs, Hi. convergens was deterred by the mealybugs themselves. There is some evidence to suggest hibiscus mealybug may have chemical defenses that make them unpalatable. Cochineal scale (Dactylopius coccus Costa; Hemiptera: Dactylopiidae) uses the anthraquinone carminic acid as a defensive compound against predators, as do several other insects that acquire it from cochineal scale (Eisner et al. 1994). Hibiscus mealybug hemolymph likely contains polyphenols like those found in cochineal scale, such as anthraquinones (Gaines et al. 2022). Based on these similarities and our observations, hibiscus mealybug may possess chemical defenses, which could explain why several predator species in our study were deterred from consuming hibiscus mealybug despite recognizing them as prey. However, this is speculative, and further research is needed to determine if hibiscus mealybug truly contains unpalatable defensive compounds.

For naturally occurring and laboratory-reared predators, our results were mixed. The coccinellids Di. austrinus and Co. septempunctata consumed few mealybug nymphs and did not consume ovisacs except in a select few instances. Co. septempunctata behaved similarly to Hi. convergens by attacking, but not consuming, many mealybug nymphs and similarly to Ch. carnea by attacking, but not consuming, mealybug adults and ovisacs. While Co. septempunctata has been documented feeding on hawthorn mealybugs (Cranshaw et al. 2000) and appeared to recognize both hibiscus mealybug nymphs and adults as potential food sources, it appears that they found them unpalatable. Di. austrinus is known as a generalist mealybug predator and can develop on multiple mealybug species (Chong et al. 2005), so it was surprising that this species fed very little on hibiscus mealybug. Again, this may point to the presence of defensive compounds.

On the other hand, two other coccinellids consumed larger numbers of hibiscus mealybug nymphs. Both Cu. coeruleus and Ha. axyridis are commonly found in Florida citrus and feed on Asian citrus psyllid (Michaud 2004; Qureshi and Stansly 2007). While neither consumed hibiscus mealybug ovisacs, their ubiquity in citrus groves and relative willingness to eat hibiscus mealybug nymphs could still make them useful generalist predators. There is also a precedent for Cu. coeruleus consuming mealybugs, and it is a known predator of Nipaecoccus nipae (CABI 2022). Promoting these predators could help control hibiscus mealybug along with Asian citrus psyllid in Florida citrus groves.

Finally, the wild caught earwig Eu. annulipes and lacewing Ceraeochrysa sp. did consume both nymphs and ovisacs at high rates in no-choice tests. Both predators have since been observed feeding on hibiscus mealybug in the field (Middleton & Diepenbrock, personal observation). While Eu. annulipes has been infrequently examined as a biological control agent, other forficulids can be important predators in orchard crops (Orpet et al. 2019), including in Spanish citrus groves where they are the primary biological control agent of aphids (Piñol et al. 2009; Romeu-Dalmau et al. 2012). We originally selected Eu. annulipes to test because they were found in cardboard tube traps used to monitor hibiscus mealybug populations (Diepenbrock, personal observation), and suspected them to be preying on the mealybugs inside. Hibiscus mealybugs have been found congregating underneath tree wraps at the base of citrus trees (Diepenbrock, observation), where they are sheltered from pesticides and possibly predators. Considering Eu. annulipes also seek out enclosed spaces and have been found underneath tree wraps, they may be particularly useful predators for hard-to-reach hibiscus mealybug in citrus. Ceraeochrysa species have been previously documented as larval predators of mealybugs and can complete development on a diet of mealybugs (Tapajós et al. 2016). Ceraeochrysa larvae are trash-carrying and cover themselves with remnants of their prey and other debris. In Florida citrus groves, we routinely saw Ceraeochrysa larvae actively feeding on mealybugs and with the remains of mealybug ovisacs and wax on their backs, similar to how Ceraeochrysa cincta (Schneider; Neuroptera: Chrysopidae) larvae were previously observed feeding on and carrying wax from the mealybug Plotococcus eugeniae (Miller & Denno) in Florida (Eisner and Silberglied 1988). From both field observations and the in-laboratory assays, Ceraeochrysa larvae appear to actively feed on multiple life stages of hibiscus mealybug.

Additional natural enemies of hibiscus mealybug also were found inside mealybug clusters collected in Florida citrus groves. Both Cr. montrouzieri and Ceraeochrysa sp. larvae were found feeding on clusters of ovisacs, further validating their potential efficacy as biological control agents. The other lacewings and predatory flies we found also have a precedent of feeding on mealybugs in the literature. The larvae of several hoverfly species in the genus Fragosa have been documented feeding on mealybugs (Rojo et al. 2003), including in Florida (Eisner and Silberglied 1988). Lacewings in the genus Sympherobius are known to consume mealybugs (Daane et al. 2008; Afifi et al. 2010) including hibiscus mealybug (Cilliers and Bedford 1978). Le. bella has been found preying on Harrisia cactus mealybug (Hypogeococcus sp.) in Puerto Rico (Triapitsyn et al. 2020), and other predatory flies from the family Chamaemyiidae (Leucopis sp.) have been found consuming Ni. viridis in South Africa (Cilliers and Bedford 1978) and Florida (Stocks 2013). An exception is An. badia larvae (Lepidoptera: Cosmopterigidae), which have not been previously documented feeding on mealybugs and instead have been found consuming plant material (Bella and Mazzeo 2006; Dawidowicz and Rozwałka 2017). However, An. badia is frequently found in close association with mealybug species (Dawidowicz and Rozwałka 2017), possibly feeding on the plant material already damaged by mealybug feeding (Bella and Mazzeo 2006). Our results demonstrate that, at least in some cases, An. badia can and will feed on the mealybugs themselves. Another species in the Anatrachyntis genus (Anatrachyntis terminella Walker; Lepidoptera: Cosmopterigidae) was recorded feeding on spider egg sacs, hollowing out cavities inside of them that are partially filled with their feces (Austin 1977). This closely aligns with what we observed in our field samples and no-choice assays, where An. badia larvae were found within hollowed-out clusters of hibiscus mealybug, held together by spun silk and partially filled with feces from the moth larvae. Futhermore, An. badia was found in all the sites sampled and had the highest abundance (n = 94) while the other two species of lepidopterans were only found in grove B at low abudance (n < 3) suggesting that An. badia is common in Florida (Table 3). To our knowledge, this is the first recorded instance of An. badia exhibiting predatory behavior on mealybugs (García Morales et al. 2016) and suggests that in previous studies where they were found in close association with mealybugs (Dawidowicz and Rozwałka 2017), they may also have been feeding on the mealybugs themselves.

Two species of parasitoids also emerged from mealybug clusters, Anagyrus dactylopii was sampled from commercial grove A and B while a species of Aprostocetus was found only in commercial grove B. Surprisingly, commercial grove B had the lowest count of ovisacs dissected but the highest abundance of parasitoids among the sites studied (331 specimens) suggesting that parasitoids can provide sufficient control to maintain mealybug populations at low to intermediate levels. At least 15 species in the genus Anagyrus are known to parasitize hibiscus mealybug (García Morales et al. 2016), and in the case of Anagyrus indicus, can significantly reduce hibiscus mealybug populations (Meyerdirk et al. 1988; Nechols and Seibert 1985; Nechols 2003). While Anagyrus dactylopii has been less frequently studied, it is an effective parasitoid of hibiscus mealybug (Sharaf and Meyerdirk 1987) and has previously been imported to Hawaii to control hibiscus mealybug (Funasaki et al. 1988). Additionally, Anagyrus dactylopii has been found to reduce hibiscus mealybug infestations on citrus and jackfruit in India (Mani et al. 2011). Tetrastichine eulophids such as Aprostocetus sp. also have been found parasitizing hibiscus mealybug (Sharaf and Meyerdirk 1987), although far less research has been conducted on them than on parasitoids in the genus Anagyrus sp. García Morales et al. (2016) listed the families Aphenelinidae (nine species), Pteromalidae (five species), and Signiphoridae (six species) as potential parasitoids of the hibiscus mealybug, however we did not find these species in our samples. Additional research to determine if more species of parasitoids are present and to what degree they impact hibiscus mealybug populations would be beneficial to inform future management practices.

Of the commercially available predators that could be released in Florida, Cr. montrouzieri appears most likely to provide control of hibiscus mealybug and can reduce severe infestations when released in CUPS production systems. Ch. carnea may also provide a measure of control by preying on mealybug nymphs but are unlikely to affect reproductive hibiscus mealybug adults. Other commercially available predators do not appear to feed on hibiscus mealybugs to any meaningful degree (García Morales et al. 2016). Releasing commercially available natural enemies could be a sustainable strategy to implement in CUPS production systems but the compatibility of pesticides with biological control agents will need to be further studied. However, the release of predators at a large scale in groves utilizing IPCs is unlikely to be economically feasible as it would be too labor intensive.

Preserving naturally occurring predators within open traditional groves may help provide control of hibiscus mealybug, in particular if Ceraeochrysa sp. and Eu. annulipes are able to persist. A suite of other predators and parasitoids prey on hibiscus mealybug and preserving them may help keep infestations at manageable levels with reduced need for insecticide applications. In production systems utilizing IPCs and CUPS, these natural enemies will most likely be physically excluded by the mesh screen. Indeed, our results showed that although the experimental CUPS production systems had the highest number of ovisacs dissected, it also had the lowest abundance of natural enemies with only two specimens of An. badia encountered. A solution that can be further investigated in IPCs is opening the bag at the bottom of the trees to allow natural enemies to enter, although there is a risk that pest arthropods also enter and infest the trees. Future work to determine if these natural enemies can effectively suppress hibiscus mealybug populations will help inform better integrated pest management approaches for hibiscus mealybug.