New Records of Parasitoids Hymenopteran ( Hymenoptera : Chalcidoidea ) Associated With Maconellicoccus hirsutus ( Hemiptera : Pseudococcidae ) in the State of São Paulo

Survey of hymenoptera parasitoids of pink-hibiscus-mealybug, Maconellicoccus hirsutus (Green, 1908) infesting Hibiscus rosa-sinensis was conducted in two municipalities of state of São Paulo, Brazil from August 2016 to August 2017. Nine species of chalcidoid wasps were obtained: Anagyrus kamali Moursi (1948); Anagyrus sp. aff. mirtesae Noyes and Menezes (2000); Anagyrus sp.; Bothriocraera bicolor Compere and Zinna (1955); Cheiloneurus sp., Gyranusoidea deionae Noyes (2000); Prochiloneurus sp. (Encyrtidae); Aprostocetus sp. (Eulophidae); and Chartocerus sp. (Signiphoridae). Anagyrus kamali and G. deionae were the most frequent species, corresponding to 67.26% and 22.62% of the parasitoids, respectively. Bothriocraera bicolor, Chartocerus and G. deionae are recorded for the first time associated with M. hirsutus. Gyranusoidea deionae and B. bicolor are recorded for the first time in Brazil. The seasonal study of M. hirsutus and its parasitoids was carried out in January to December 2017on 16 hibiscus plants, aligned in the hedge in the experimental area of the FCAV/UNESP in Jaboticabal, SP. For this purpose, ovisacs, nymphs and adults of mealybug were counting on infested plants and the adult parasitoids collected in Möerick traps. Maconellicoccus hirsutus was observed during the periods of January-March and October-December, with the highest infestation in January and February. In the traps, a total of 92 specimens of pink-mealybug parasitoids were obtained: 65% belonging to A. kamali and 35% to G. deionae.


Introduction
The pink-hibiscus-mealybug (PHM), Maconellicoccus hirsutus (Green, 1908) (Hemiptera: Pseudococcidae), is a polyphagous species reported on more them 300 species of host plants, distributed in 78 families, mainly in the tropical and subtropical regions of the world (García Morales et al., 2018).
The management of M. hirsutus might be performed in different ways, including chemical control indicated mainly for high infestations in restricted environments, such as nursery planting, where infested plants do not resist the presence of the pest (Chong et al., 2015).However, the waxy cover, eggs inserted in a filamentous secretion (ovisac), and habit of nymphs and adult females to settle in cryptic parts of the host plant, protects them from contact with insecticidal sprays (Kairo et al., 2000).
Biological control has been extensively used for this species.Worldwide, 85 natural enemies have been reported that are associated with M. hirsutus, 39 are hymenopterous parasitoids (Hymenoptera: Encyrtidae) and 46 predators, predominantly distributed in the families of Coccinellidae (Coleoptera) and Chrysopidae (Neuroptera) (Chong et al., 2015, Peronti et al., 2016).
However, the complex of natural enemies associated with an insect pest can be variable between different zoogeographic regions or even in different parts of the same country, demonstrating the importance of regional surveys.Goolsby et al. (2002) -Júnior et al., 2013, Peronti et al., 2016).
A. kamali and G. indica are important in biological control programs of PHM, mainly in the South of North America and the Caribbean region (Kairo et al., 2000).In states of California, Florida, Hawaii (USA) and Mexico, biological control programs using these natural enemies were successfully achieved up to 95% reduction of M. hirsutus population (Roltsch et al., 2006).
In Roraima, northern Brazil Negrini et al. (2018) studied the population fluctuations of M. hirsutus and its natural enemies in a sugar-apple orchard, Annona squamosa L. (Anonnaceae) and verified highest infestation occurred in August and February-March and the average parasitism by A. kamali in fruits of the 50%, with highest rates in periods of greatest infestation by M. hirsutus.
Therefore, the objective of this work was to increase the knowledge about the hymenopterous parasitoids associated to M. hirsutus in the state of São Paulo and study of the seasonality of M. hirsutus and its parasitoids on Hibiscus rosa-sinenses in an experimental area in the municipality of Jaboticabal, SP.

Survey of Natural Enemies
The branches, leaves and flowers of Hibiscus rosa-sinensis infested by M. hirsutus were collected in public and private gardens in the municipalites of Campinas, Jaboticabal and São Carlos, SP, Brazil and transported to the Laboratory of Biosystematics of Hemipteran (LABHEM) of the Plant Protection Department of the Universidade Estadual Paulista "Julio de Mesquita Filho"-Campus Jaboticabal, from August 2016 to August 2017.Sporadic samples were also obtained in Jales, Ribeirão Preto, São Paulo, Vinhedo and Votuporanga, SP.
The collected part of the specimens of M. hirsutus was stored in vials containing 70% alcohol for posterior montage and identification.Other parts were placed in glass tubes sealed with paper film under controlled conditions (25±2 °C, photoperiod of 12 hours and UR 65±5%) in BOD incubator for 25 days, according to methodology adapted from Prado et al. (2015).At each 48 hour intervals, the emerged parasitoids were transferred to 2 ml eppendorf microtubes filled in 70% alcohol.
The mealybugs were slided-mounted following the methodology described by Granara de Willink (1990) and identified according morphological characteristics of the adult female as described by (Miller, 1999;Miller et al., 2011).
Most of hymenopterous parasitoids were prepared in double-mount (Hanson & Gauld, 2006).Species smaller than 0.7 mm were mounted on permanent slides following the technique of Querino and Zucchi (2011).Subsequently, the parasitoids were identified under optical stereoscopic microscope at the genus or species level, according to the following works: Fernández and Sharkey (2006) and Hanson and Gauld (2006) for identificati (1997); an and Hayat Specimens (CRIA) of of Entomo

Season
The sampl environme December In the sam plants and counted un for identifi

Season
The pink-h highest inf evaluated temperatur experimen In the trap months of species ha mealybug; parasitoids (Figure 3).Gyranusoidea deione is a Neotropical species of uncertain origin, registered only in Costa Rica (Noyes, 2000).It is very similar to G. indica, differing mainly in having an intense dark spot that connects the torulus and eye, the mid coxa is concolorous with the hind coxa, in addition to the length of the gonostylus 0.25x as long as the ovipositor.In G. indica the spot between the torulus and the eyes is conspicuous and dark brown; the hind coxae is pale yellow contrasting with brown mid coxa; and the length of the gonostylus 0.25x as less as the ovipositor (Noyes, 2000).

Eulophidae
Eulophidae are parasitoids of a great diversity of arthropods, being found parasitizing from arachnids, nematodes and distinct species of insects during various stages of development (La Salle, 1994).There are few associations between eulophids with mealybugs.Most species have been associated as primary or secondary parasitoids of coccids and other 41 families of arthropods (La Salle, 1994;Peck, 1963).
Aprostocetus Westwood, 1833 is a very complex genus, with more than 800 species registered around the world (La Salle, 1994).Aprostocetus minutus (Howard, 1881) is the species with the highest number of hosts belonging to the family Pseudococcidae, being associated with six genera in the world (La Salle, 2006;Noyes, 2018).The species reported in Brazil are not associated with this family of scale insects, representing 15 species in the country, but the genus Aprostocetus has already been cited in the literature associated with Pseudococcidae in Brazil (Lopes et al., 2017;Chong et al., 2015).

General Discussion
Nine species of hymenopterous parasitoids were recorded to be associated with M. hirsutus in the state of São Paulo distributed into seven genera.According to the literature, species of Anagyrus, Gyranusoidea, Bothriocraera have been mentioned preferentially as primary parasitoids of mealybugs and those of the genera Chartocerus sp., Cheiloneurus sp. and Prochiloneurus sp. as secondary parasitoids.Species of the genus Aprostocetus have been related as primary and secondary parasitoids (Table 2).Noyes, 1994;Moursi, 1948;Miller, 1999.Anagyrus sp.aff.mirtesae Unknown Noyes, 2000;Sánchez-García, 2016.

Prochiloneurus
Hyperparasitoids of Hemiptera (Coccidae, Pseudococcidae and and primary parasitoid of Chalcidoidea (Encyrtidae).Noyes, 1984;Prinsloo, 1983;Triapitsyn, 2014 Anagyrus was the genus with the largest number of specimens, A. kamali, A. sp.aff.mirtesae and Anagyrus sp.; Species of this genus are known for their high specificity to mealybugs.Of the nine registered hosts for A. kamali, eight are from pseudococcids (Noyes, 2018).This exotic hymenopteran was probably introduced in Brazil together with the pink-hibiscus-mealybug, both recorded for this country in the same year, in Boa Vista, Roraima (Marsaro-Junior et al., 2013).For A. mirtesae, previously known to Costa Rica and Mexico, no host has been known until now (Noyes, 2000;Sánchez-García, 2016).
For Gyranusoidea deionae, the second species with the largest number of specimens was reported as a first host of it in this study.This species is extremely similar to G. indica which as obtained from M. hirsutus by Peronti et al. (2016) in the state of São Paulo.However, it was not found during this survey.Due to the great similarity of them morphometric and molecular studies would be required to verify the status and distribution of both.
For the Neotropical region, 19 species of parasitoids had previously been associated with M. hirsutus (Chong et al., 2015;Culik et al., 2013).Adding the four species here reported for the first time to this region, 23 species are now known.In general, Anagyrus and Gyranusoidea include species better distributed in the Australian and Oriental regions, except for a few species such as Anagyrus sp.aff.mirtesae and G. deionae, which are only registered in Costa Rica, Mexico and Brazil.
The number of species of hymenopterous parasitoids associated with mealybugs has been increased in recent years, however the records, have been done to isolated species.Studies that include surveys of species in a given region are less frequent (Chong et al., 2015;Culik, et al., 2013;Mani et al., 1987).Surveys of hymenopterous parasitoids associated with M. hirsutus were conducted in India by Mani et al. (1987) and Michaud & Evans, (2018) in Puerto Rico.In India: A. dactylopii, Anagyrus mirzai Agarwal and Alam, 1959 (= Gyranusoidea mirzai) and Alamella flava Agarwal, 1966 (Encyrtidae), Allotropa sp.Near A. japonica Ashmead, 1904 (Platygastridae), Leptopilina sp.(Figitidae) and Chartocerus sp.Near C. walkeri Hayat, 1970, (Signiphoridae) were reported and in Puerto Rico, A. kamali, G. indica, Acerophagus nubilipennis Dozier, 1926, C. inimicus and A. minutus.Of the six species obtained in India, two are included in common genera to those found in this study, Anagyrus spp.and Chartocerus sp.Four of the five species associated with PHM in Puerto Rico belong to genres common to those species found in this study with A. kamali representing 70% of the number of parasitoids obtained.
In the municipality of Jaboticabal-SP, the population peaks of M. hirsutus in the years 2016-2017 occurred in January and February, where temperatures were higher.These results corroborate with those obtained by Negrini et al. (2018), demonstrating that in the months of May-July there is no presence of mealybug in the field, but in the months that followed, in the periods of spring and summer, February and end of September, respectively, the greater amount of mealybugs occur and their re-establishment in the field, also resulted in the appearance of the natural enemies.Negrini et al. (2018) evaluated the number of M. hirsutus mummies, corresponding to parasitized mealybugs, in A. squamosa fruits.The highest pupal average was obtained in September, February and March, being the period corresponding to the highest population peaks of PHM, all of A. kamali.In the present study, hymenopteran parasitoid species were collected from Möericke traps and two species known to parasite PHM were captured (A.kamali and G. deionae).The seasonality of A. kamali in relation to population peaks of M. hirsutus was similar in both studies.This survey contributed to report new species of natural enemies associated with M. hirsutus in the world and in Brazil particularly in addition, the study demonstrated that this exotic pest is being naturally controlled in the state of São Paulo by a complex of hymenoptera parasitoids especially A. kamali corresponding to more than 65% of the parasitoids obtained by manual collection of parasitized mealybugs and traps.

Table 1 .
Hibiscus s by species y of Natural En f 168 specime distributed in n Chartocerus an idea deionae a f São Paulo (F 1. Parasitoids (A) Anagyrus us sp.aff.mirt e presented the A. kamali and species with a nemies ens of parasit nine species: 7 nd G. deiona are recorded fo Figure 1).associatedwit kamali; (B) Gy tesae; (F) Both e highest num d G. deionae small number

Table 2 .
Species by family, associations and literature pertinent to their hosts.* numbers in parentheses are corresponding to the number of host families of the parasitoid .