A Survey in Hawaii for Parasitoids of Citrus Whiteflies (Hemiptera: Aleyrodidae), for Introduction into Greece

Simple Summary The orange spiny whitefly, Aleurocanthus spiniferus, has invaded Greece and expanded its distribution in other countries in the European Union since 2008. It is a polyphagous pest that includes several highly important crop plants, such as citrus. Damage symptoms and effects on plants are identical to those of the citrus blackfly, Aleurocanthus woglumi (both Hemiptera: Aleyrodidae). The species also share a complex of natural enemies that were successfully introduced to Hawaii during 1974–1998. A short expedition to the islands of Oahu, Hawaii, and Kauai was conducted to retrieve the prominent natural enemies for introduction into Greece. Hawaii was chosen because it does not have citrus diseases and because of the existence of connections to facilitate research and export permits. Infested leaves were shipped to a quarantine facility in Greece for parasitoid emergence and evaluation. The identity of emerged parasitoids and host testing on the orange-spiny whitefly Greece strain were conducted. Only one parasitoid was abundant, characterized using molecular analysis as Encarsis perplexa. A summary of infestation records, parasitism rates, and localities on the Hawaiian Islands has been reported here for the first time since the release of parasitoids. Results showed that the infestations of citrus trees were minimal on the islands of Hawaii and Oahu, primarily on pummelo and sweet orange. Citrus whiteflies were not detected on the island of Kauai during this survey. E. perplexa had parasitism rates ranging from 0 to 28% on the island of Hawaii and 11 to 65% on the island of Oahu. A starter colony of the parasitoids has been colonized in the Greece Quarantine Facility for evaluation. This was the first field survey of Hawaii since the introduction and release of citrus whitefly natural enemies. Further surveys should be repeated in different countries to eliminate the risk of disease introduction. Abstract Whitefly species of Aleurocanthus spiniferus (Quaintance) and A. woglumi Ashby (Hemiptera: Aleyrodidae) are serious pests of citrus and other important fruit crops. The problem of citrus has initiated the successful introduction of several natural enemies for biocontrol programs in Hawaii and many other countries. Here, we summarized the history of infestation and biocontrol efforts of the two whiteflies in Hawaii for possible parasitoid importation into Greece. Two Platygasteridae (Amitus hesperidum Silvestri, A. spiniferus (Brethes), and three Aphelinidae (Encarsia clypealis (Silvestri), E. smithi (Silvestri), E. perplexa Huang, and Polaszek) were released in Hawaii for biocontrol of the citrus whiteflies during the period 1974–1999. The aphelinid Cales noacki Howard, purposely released for Aleurothrixus flococcus (Maskell) in 1982, was also reported to attack other whiteflies, including Aleurocanthus species, on citrus. An additional aphelinid parasitoid, Encarsia nipponica Silvestri, native to Japan and China, was accidentally introduced and found to attack both citrus whiteflies on the islands. Since the colonization of introduced parasitoids in infested fields on four Hawaiian Islands, no survey has been conducted to evaluate their potential impact. We conducted two short surveys during September–November 2022 on the islands of Kauai, Hawaii, and Oahu to introduce the dominant parasitoids to Greece for the biocontrol of A. spiniferus. Results showed that the infestation level was very low on Kauai, Hawaii, and Oahu Islands, with a mean infestation level range of 1.4–3.1 on Hawaii and Oahu Islands, mostly on pummelo and sweet orange, with no detection on the island of Kauai. The dominant parasitoid was characterized as Encarsia perplexa, using molecular analysis. Its parasitism rates ranged from 0 to 28% on the island of Hawaii and 11 to 65% on the island of Oahu. Emerged parasitoids have been reared in Greece for evaluation. This was the first field survey of Hawaii since the introduction and release of citrus whitefly natural enemies.


Simple Summary:
The orange spiny whitefly, Aleurocanthus spiniferus, has invaded Greece and expanded its distribution in other countries in the European Union since 2008.It is a polyphagous pest that includes several highly important crop plants, such as citrus.Damage symptoms and effects on plants are identical to those of the citrus blackfly, Aleurocanthus woglumi (both Hemiptera: Aleyrodidae).The species also share a complex of natural enemies that were successfully introduced to Hawaii during 1974-1998.A short expedition to the islands of Oahu, Hawaii, and Kauai was conducted to retrieve the prominent natural enemies for introduction into Greece.Hawaii was chosen because it does not have citrus diseases and because of the existence of connections to facilitate research and export permits.Infested leaves were shipped to a quarantine facility in Greece for parasitoid emergence and evaluation.The identity of emerged parasitoids and host testing on the orange-spiny whitefly Greece strain were conducted.Only one parasitoid was abundant, characterized using molecular analysis as Encarsis perplexa.A summary of infestation records, parasitism rates, and localities on the Hawaiian Islands has been reported here for the first time since the release of parasitoids.Results showed that the infestations of citrus trees were minimal on the islands of Hawaii and Oahu, primarily on pummelo and sweet orange.Citrus whiteflies were not detected on the island of Kauai during this survey.E. perplexa had parasitism rates ranging from 0 to 28% on the island of Hawaii and 11 to 65% on the island of Oahu.A starter colony of the parasitoids has been colonized in the Greece Quarantine Facility for evaluation.This was the first field survey of Hawaii since the introduction and release of citrus whitefly natural enemies.Further surveys should be repeated in different countries to eliminate the risk of disease introduction.
Abstract: Whitefly species of Aleurocanthus spiniferus (Quaintance) and A. woglumi Ashby (Hemiptera: Aleyrodidae) are serious pests of citrus and other important fruit crops.The problem of citrus has initiated the successful introduction of several natural enemies for biocontrol programs in Hawaii and many other countries.Here, we summarized the history of infestation and biocontrol efforts of the two whiteflies in Hawaii for possible parasitoid importation into Greece.Two Platygasteridae (Amitus hesperidum Silvestri, A. spiniferus (Brethes), and three Aphelinidae (Encarsia clypealis (Silvestri), E. smithi (Silvestri), E. perplexa Huang, and Polaszek) were released in Hawaii for biocontrol of the citrus whiteflies during the period 1974-1999.The aphelinid Cales noacki Howard, purposely released for Aleurothrixus flococcus (Maskell) in 1982, was also reported to attack other whiteflies, including Aleurocanthus species, on citrus.An additional aphelinid parasitoid, Encarsia nipponica Silvestri, native to Japan and China, was accidentally introduced and found to attack both citrus whiteflies on the islands.Since the colonization of introduced parasitoids in infested fields on four Hawaiian Islands, no survey has been conducted to evaluate their potential impact.We conducted two short surveys during September-November 2022 on the islands of Kauai, Hawaii, and Oahu to introduce the dominant parasitoids to Greece for the biocontrol of A. spiniferus.Results showed that the infestation level was very low on Kauai, Hawaii, and Oahu Islands, with a mean infestation level range of 1.4-3.1

Introduction
The orange spiny whitefly, Aleurocanthus spiniferus (Quaintance), and citrus blackfly, A. woglumi Ashby (Hemiptera: Aleyrodidae), are two of the most important and hazardous citrus pests [1,2].Both species are regulated as quarantine pests for the European Union [3].Aleurocanthus woglumi has not invaded Europe yet.However, climatic modeling predicts that areas in the Mediterranean basin are suitable for establishment [4].Aleurocanthus spiniferus has already invaded Europe, with the first record in Italy in 2008 [5].Since then, its distribution in the European Union has expanded, including Albania [6], Croatia [7], Greece [1], and Montenegro [8].Both species cause similar symptoms to the infested host plants.In Hawaii, during a heavily infested period in 1998, infested trees were not fruiting, with a range of 50->600 citrus blackfly pupae per infested leaf ([2], Ramadan, Hawaii Department of Agriculture (HDOA) unpublished record).The main hosts are citrus species, but they are quite polyphagous, with a wide host range that includes mangoes and coffee [9].Direct damage is caused by nymphs that suck the sap from the leaves, causing a lack of nutrients and water.Additionally, nymphs excrete honeydew drops that fall on the leaves, where the growth of sooty mold is promoted, which eventually covers the whole upper surface of the infested leaves.Both A. spiniferus and A. woglumi originate from Southeastern Asia [4,10].In their native area, they are not considered major pests; however, during the 20th century, both species invaded other parts of the world, causing substantial damage, mainly to citrus crops [2][3][4].Following their accidental introduction to new areas, biological control programs were initiated to mitigate the damage caused by the outbreaks of the two whiteflies [2,[10][11][12][13][14].
In the early seventies, A. spiniferus was introduced in Hawaii on rose foliage in Honolulu, Oahu Island, in 1974.Subsequent surveys discovered it on navel orange, lime, tangerine, and pear, but infestations were reportedly low [15].Two decades later, A. woglumi was also found being introduced on the main Hawaiian Islands [15,16].In both cases, the introduction of citrus whitefly species followed the introduction of biological control agents to compact their population outbreaks.Specifically, the parasitoid Encarsia smithi Silvestri and E. clypealis (Silvestri) (Hymenoptera: Aphelinidae) were introduced in the islands of Oahu and Hawaii [17] to control A. spiniferus, and E. perplexa Huang and Polaszek (Hymenoptera, Aphelinidae) (misidentified at that time as Encarsia opulenta Silvestri) and Amitus hesperidum Silvestri (Hymenoptera: Platygastridae) were introduced to control A. woglumi [16], Table 1.Encarsia smithi was introduced to the island of Oahu from Japan in 1974, among three other Aphelinid species that were found to naturally parasitize A. spiniferus in Oahu.Encarsia smithi was the most important species, leading to a sufficient reduction of the A. spiniferus population [17].It was also reported on A. woglumi (Table 1).In 1998, exploratory investigations were carried out in Central America in Guatemala, and two parasitoid species, E. perplexa and A. hesperidum, were introduced to Hawaii.The wasps were mass-reared on Oahu and released on other islands (Table 1).The two wasp species managed to disperse naturally and successfully controlled the population of A. woglumi in all releasing sites (Table 1) [18].Another parasitoid, E. nipponica Silvestri, native to Japan and China, was discovered accidentally as a parasitoid of both whiteflies with low parasitism ranging from 0 to 11.5% (n = 103 infested leaves, Ramadan unpublished data).
Similarly, the aphelind parasitoid, Cales noacki Howard, was released in 1981 for biological control of Aleurothrixus floccosus (Maskell), subsequently reported to attack Aleurocanthus species on citrus [19], HDOA records.There has been no field evaluation since the introduction and release of all the parasitoids, but citrus growers on the islands were content, and less chemical control was used in their fields [2].The recent invasion of A. spiniferus in the European Union and Greece, in particular, has caused the initiation of a new biological control program against this pest by introducing exotic natural enemies.Moreover, since the introduction of these parasitoids in Hawaii, no surveys have been conducted to determine the status of A. woglumi or A. spiniferus in the islands and the presence and abundance of their introduced parasitoids.Thus, an exploratory investigation on the Hawaiian Islands was performed to investigate the current parasitism rate of A. woglumi and to identify the parasitoid assembly species that were present in the populations of the pests.The results of those short surveys will provide crucial information to examine the possibility of a successful introduction of those parasitoids, either in Greece or in any other area where those serious pests have been established.

Collection and Emergence of Parasitoids
Two surveys were performed in September and November 2022 on the islands of Hawaii, Kauai, and Oahu (Figure 1).A total of 62 sites located on the three Hawaiian Islands were surveyed (Hawaii Island with the GPS coordinates of 19       2 and 3.

Identification of the Parasitoids
The identification of emergent parasitoids was based on morphological characteristics and molecular analyses.Morphological identification was performed according to appropriate keys and illustrations [20][21][22][23].For distinguishing Amitus hesperidum: The female is shiny black (0.75 mm long).The female′s antenna is tensegmented, with the last three segments forming a club.The male is like the female, with a filiform ten-segmented antenna and curved scape.A lateral plate-like process on the  2 and 3.  Host plants (mainly citrus trees) were macroscopically surveyed for citrus whitefly infestation, and infested leaves were collected (Tables 2 and 3).Citrus whiteflies have been very rare to be found in Hawaii in recent years.Sites were selected as orchards of citrus, trees in agriculture experimental stations, and local residential homes were examined with the permission of landlords.Sites were selected randomly for the survey team to enter property everywhere on the visited islands.Based on the presence of citrus trees, the survey team searched the trees for infestations and obtained permission to clip infested branches or leaves.Leaves with mature whiteflies were picked, not those with only eggs or small nymphs that would not produce parasitoids.
The infested leaves were placed in paper envelopes, which were sealed (AJM paper grocery, lunch bags, USA, of different sizes as needed for holding infested leaves).Plastic bags are not suitable for live insects, https://www.gofoodservice.com/brand/ajm(accessed on 10 September 2023).Infested leaves were picked according to the stage of the nymphs of the citrus blackfly.Leaves that carried all the stages and older nymphs were preferred.Leaves with mature nymphs (third and fourth nymphs) were preferred for collection.Leaves with eggs-second nymphs-were not collected since parasitoids from such leaves may not develop in the holding containers.
At the end of each collection day, all envelopes with leaf samples were transported to the insectary of the Hawaii Department of Agriculture, Plant Pest Control Branch in Honolulu.In the insectary, the leaves were carefully removed from the envelopes and placed in 70 mesh clear vinyl nylon screen collapsible lightweight aluminum cages (30 × 30 × 60 cm) appropriate for parasitic wasps emergence (https://www.bioquip.com(accessed on 20 October 2023)).Minute honey drops were placed on the inner surface of the mesh as food for adult wasps.We used smeared drops of SUE BEE ® SPUN ® honey on the inside cage sides and top for adult parasitoid feeding as indicated by HDOA insect rearing (https://siouxhoney.com/sue-bee-spun-honey/ (accessed on 20 July 2022)).Emerged adult parasitoids were aspirated and collected in falcon vials with tiny honey drops (Falcon 50 mL Conical Centrifuge Tubes; Fisher Scientific: A Thermo Fisher Scientific Brand (https://www.fishersci.com/shop/products/falcon-50ml-conical-centrifuge-tubes(accessed on 15 September 2023)).

Identification of the Parasitoids
The identification of emergent parasitoids was based on morphological characteristics and molecular analyses.Morphological identification was performed according to appropriate keys and illustrations [20][21][22][23].For distinguishing Amitus hesperidum: The female is shiny black (0.75 mm long).The female s antenna is ten-segmented, with the last three segments forming a club.The male is like the female, with a filiform ten-segmented antenna and curved scape.A lateral plate-like process on the male fourth antennal segment is characteristic of the species, Figure 2D [20,21,23].For identification of Encarsia perplexa, the mid lobe of the mesoscutum is dark, and T1 and T2 of the gaster are largely pale (Figure 2C).The male head is like that of the female.The mesosoma is orange-yellow except for the pronotum.The anterior half of the mid lobe of the mesoscutum, the propodeum, and the petiole are dark brown.The gaster is brown to dark (Figure 2C).Other characteristics for the differentiation of closely related species are explained in [22].The specimens were prepared for slide mounting as described in slide preparation of chalcidoids by Noyes [24][25][26].

Results and Discussion
Survey results indicated that A. woglumi populations are rare in the Hawaiian Islands.Out of the 62 locations inspected for A. woglumi populations, its presence was found in only 11 sites (Figure 1).On Kauai Island, no infestation was found, and on Hawaii Island, A. woglumi populations were found in just 3 sites out of the 23 inspected.Moreover, the infestation rate was quite low (level of infestation 1.37-3.14),and only isolated citrus trees either on Oahu or in the Hawaii Islands were found with a large number of infested leaves (≥10 leaves/tree).Throughout the survey, populations of other Aleyrodidae were very low, and only a few isolated populations of Aleurothrixus floccosus (Maskell) and Aleurodicus dispersus Russell were found.
Parasitism of A. woglumi nymphs was much higher on Oahu than on Hawaii Island.Parasitism rates ranged from 0 to 28% on Hawaii Island and 11 to 65% on Oahu Island (Tables 2 and 3).
Based on the morphological and molecular data, the emerging parasitoids were identified as E. perplexa.A few emerging parasitoids were identified as Amitus spiniferus Brethes, a parasitoid introduced to Hawaii for A. floccosus.
Specifically, the molecular comparison via the NCBI (National Center for Biotechnology Information) database resulted in high similarity rates, suggesting that the analyzed parasitoids undoubtedly belong to the genus Encarsia.Most of the consensus sequence produced belongs to A. hesperidum.Our survey results were similar to those of E. perplexa, at a range of 98.6 to 99.5%.The NCBI contained only limited data for the genus Amitus for both genes, a fact that limited the determination of some individuals to the genus level.Sequencing data provided by the USDA supports the claim that these parasitoids are highly similar to the species Amitus spiniferus, much more so than to A. hesperidum.
Our survey results confirm that A. woglumi on the Hawaiian Islands is effectively controlled by the introduced natural enemies and that E. perplexa is the dominant parasitoid species [2].We were not able to find A. hesperidum either due to competition with E. perplexa or because its population is at very low levels, not being detectable with our sampling effort during this short survey.In other locations where both E. perplexa and A. hesperidum have been released to control A. woglumi, soon after the reduction in the infestation, E. perplexa had become the dominant species.This is mainly due to its longer lifespan compared to A. hesperidum.The latter is considered an effective parasitoid at high densities of its host, whereas E. perplexa is more efficient at low host densities [11].
In 1997, the infestation by A. woglumi was so severe on Oahu Island that every citrus tree, including oranges, lemons, and pummelo, had a range of 50->600 citrus blackfly pupae/leaf (Figure 2A).In 1998, all citrus trees on Oahu (urban trees and orchards) had been reduced to no fruiting from the high infestation.There were complaints from citrus growers on the Hawaii and Kauai Islands requesting parasitoid introductions.Recently, there have been no reports of damage incurred by the pest or reports of growers or stakeholders having issues with citrus blackflies.
Non-citrus trees and ornamental shrubs (orange jasmine, Murraya paniculate (L.), Rutaceae) were also utilized for oviposition but were not normally affected by this whitefly.The mean number of egg masses/leaflet was 16.4 ± 2.8 (n = 25, during June 1997, Oahu Island) and 22.4 ± 3.1 eggs/mass (n = 22).Citrus blackfly eggs hatched with no development beyond the first nymph on non-citrus trees.
From the adult parasitoids that emerged either in Hawaii and shipped into Greece or emerged as adults in the quarantine facility of Benaki Phytopathological Institute in Emerged adult parasitoids were aspirated and collected in falcon vials with tiny honey drops (Falcon 50 mL Conical Centrifuge Tubes; Fisher Scientific: A Thermo Fisher Scientific Brand (https://www.fishersci.com/shop/products/falcon-50ml-conicalcentrifuge-tubes(accessed on 15 September 2023)).Emerged parasitoids and remaining infested leaves were shipped to Greece and placed in the containment facility in the biosecurity greenhouse at the Scientific Directorate of Entomology and Agricultural Zoology, Benaki Phytopathological Institute (38°04′52.0″N 23°48′47.9″E).Emerged parasitoids were placed inside mesh cages with bitter orange Citrus aurantium L. (Rutaceae) seedlings infested by A. spiniferus in the biosecurity greenhouse under controlled environmental conditions of 25 °C ± 2 °C, R.H. 60-70%, and natural daylight photoperiod (daylight hours 6:40 a.m.-5:30 p.m.).Citrus aurantium seedlings were in mesh cages (45 × 45 × 45 cm), which were put inside larger ones (60 × 60 × 60 cm, 70 mesh).Polyester Chiffon white breathable fabric was used to cover cages for rearing delicate encyrtid-size parasitoids (https://www.moodfabrics.com/fashion-fabrics/polyester/chiffon(accessed on 10 October 2023)).

Figure 1 .
Figure 1.Sampling locations for Aleurocanthus woglumi and its parasitoids in the Hawaiian Islands (red circle).Sampling locations with GPS coordinates are shown in Tables2 and 3.

Figure 1 .
Figure 1.Sampling locations for Aleurocanthus woglumi and its parasitoids in the Hawaiian Islands (red circle).Sampling locations with GPS coordinates are shown in Tables2 and 3.

Insects 2023 , 16 Figure 2 .
Figure 2. (A) Citrus blackflies, Alerocanthus woglumi (arrow showing unparasitized pupa and egg spirals); (B) orange spiny whiteflies, A. spiniferus, and their parasitoid assembly established in Hawaii.The pupal stage of the orange spiny whitefly with the white wax fringe that surrounds its margins is twice as large as the orange spiny whitefly; (C) Encaria perplexa, male and female, introduced from Guatemala in 1998; (D) Amitus hesperidum, introduced from Guatemala in 1998; (E) Citrus blackfly parasitized pupae, arrows showing parasitoid′s circular exit holes.The oval-shaped pupa is black and convex and has dark dorsal spines.The marginal wax tubes produce a cottony

Figure 2 .
Figure 2. (A) Citrus blackflies, Alerocanthus woglumi (arrow showing unparasitized pupa and egg spirals); (B) orange spiny whiteflies, A. spiniferus, and their parasitoid assembly established in Hawaii.The pupal stage of the orange spiny whitefly with the white wax fringe that surrounds its margins is twice as large as the orange spiny whitefly; (C) Encaria perplexa, male and female, introduced from Guatemala in 1998; (D) Amitus hesperidum, introduced from Guatemala in 1998; (E) Citrus blackfly parasitized pupae, arrows showing parasitoid s circular exit holes.The oval-shaped pupa is black and convex and has dark dorsal spines.The marginal wax tubes produce a cottony fringe on the pupal margin; (F) Encarsia nipponica adventive; (G) Encarsia smithi, introduced from Japan in 1975 and established on Oahu.

Table 2 .
Infestation rates and parasitization of A. woglumi infesting Citrus species on Oahu Island during September-November 2022.a residential house, roadside, highway service area, farmland, research station, community garden, cemetery, park, and church.

Table 3 .
Infestation rates and parasitization of A. woglumi infesting Citrus species on Hawaii Island during October 2022.a residential house, roadside, highway service area, farmland, research station, community garden, cemetery, park, and church.