Complex of Natural Enemies Associated With Scale Insects ( Hemiptera : Coccomorpha ) on Sugarcane in Brazil

A survey of the natural enemies of Aclerda takahashii (Kuwana, 1932) (Hemiptera: Aclerdidae) and Saccharicoccus sacchari (Cockerell, 1895) (Hemiptera: Pseudococcidae) was carried out in the state of São Paulo, Brazil. A total of ten associations were recorded, six parasitoid-host and four predator-prey, which three are new: parasitoids of the genus Eurytoma (Hymenoptera: Eurytomidae) and Hambletonia (Hymenoptera: Encyrtidae) associated with A. takahashii and S. sacchari, respectively; and the predator of the genus Diadiplosis (Diptera: Cecidomyiidae) with a S. sacchari. Anagyrus saccharicola Timberlake, 1932 and Mariola flava Noyes, 1980 (Hymenoptera: Encyrtidae) are reported for the first time in Brazil. Keyswords: ecological interactions, parasitoids, predators, São Paulo state

female and 4-5 for males.The occurrence of polymorphism in males of this mealybug was reported by Hafez and Salama (1967), with apterous, winged and intermediate forms.In laboratory conditions in Egypt, only sexual reproduction was observed, with four to five generations per year (Hafez & Salama, 1969).For A. takahashii this information is unknown.
In Brazil, sugarcane scale insects, especially S. sacchari, have been considered as emergent pests, since sugarcane infestation by this mealybug has been reported frequently by producers (Personal communication, Modesto Barreto).However, the increase of population of this mealybug may be related to the changes in the management of the sugarcane fields in the state of São Paulo.Burning the mature crops of sugarcane was practiced for centuries in Brazil but, currently this management has been substituted by the unburned harvest.In addition, a straw cover, consisting by dried leaves and stalks, probably also contributes for the presence of these insects in the field.
Sacharicoccus sachari and A. takahashii settle under overlapping leaf sheaths just below the nodes, which hinders the chemical control (Alam, 1972;Stocks, 2016).In underground S. sacchari infests roots and newly planted seed cane facilitating their spread throughout the area during planting (Beardsley, 1960).The use of insecticides in seed is possible, but considered economically impracticable.Effective control could be achieved through cultural practices and biological control (Rajendra, 1974).Associated with S. sacchari and A. takahashii, 37 species of predators and 45 species of parasitoids (Appendix A) were registered globally.These predators are larvae or adults of Coccinellidae and Staphylinidae (Coleoptera), larvae of Chrysopidae (Neuroptera), Cecidomyiidae, Drosophilidae and Phoridae (Diptera), earwigs of families Forficulidae and Labiduridae (Dermaptera), bedbugs of the families Anthocoridae and Geocoridae (Hemiptera) and microlepdopterous of the families Pyralidae and Momphidae (Lepidoptera), commonly found feeding on eggs, nymphs and adults of coccoids.Parasitoids belong to the families Aphelinidae, Encyrtidae, Pteromalidae, and Signiphoridae (Hymenoptera: Chalcidoidea) are among the most important groups of natural enemies of these scale insects (Noyes, 2017) (Appendix A).In Brazil, natural enemies associated with sugarcane scale insects were recorded mainly by Lima (1935), andDe Santis (1972).
The survey of natural enemies, especially of well-established native or exotic species, associated with insects of agricultural importance in a given region is an important instrument for IPM (Integrated Pest Management).In state of São Paulo despite the apparent increase in the populations of scale insects infesting the crop the possible natural enemies associated with them are unknown.The objective of this study was to survey species of natural enemies associated with S. sacchari and A. takahashii, in São Paulo sugarcane plantations; checking the occurrence of natural enemy known in other countries, and to reporting new associations.
The scale insects and their respective natural enemies were randomically collected from infested sugarcane plants: (A) in small clumps located in backyards and farms, and in the (B) sugarcane field edges.

Obtaining Natural Enemies
Infested nodes were inspected visually mainly in looking for immature predators.Coccinellids and dipterous larvae obtained were placed in transparent plastic pots (16 × 12 × 5 cm in size), covered with muslin netting and fed with nymphs of the scale insect in which they were found associated on the sugarcane field.The adult predators emerged or collected manually on the colonies of scale insects were posteriorly pinned.
Parasitoids were obtained from ten adults of each scale insects sampled.The scale insects were placed in glass tubes partially covered with laminated paper and closed with plastic film, according to methodology adapted from Prado, Alvarenga, and Santa-Cecília (2015).At 48 h the emerging adult parasitoids were transferred to 2 mL eppendorf and preserved in 100% ethanol.Subsequently, they were fixed on entomological pins.The procedures were performed under controlled conditions (25±2 °C, photoperiod of 12 h and UR 65±5%), in B.O.D.

Slide-Mounted Procediments for the Scale Insects
Some specimens of coccoids of each sample previously preserved in 75% ethanol were mounted on microscopic slides following the method described by Granara de Willink (1996).Posteriorly, they were identified according to morphological characteristics of the adult female as described by Mcconnell (1954) for Aclerdidae and Williams and Granara de Willink (1992) for Pseudococcidae.

Identification
The scale insects collected on nods of sugarcane plants in the state of São Paulo were confirmed as S. sacchari and A. takahashii.A total of 394 specimens of natural enemies associated with them were collected in the 17 sampled sites: five per both species, seven to S. sacchari and five to A. takahashii (Appendix B).The natural enemies complex associated with the sugarcane scale insects obtained includes six species of hymenopterous parasitoids and four species of predators (Table 1).(Alam, 1972;Gordon, 1985;Sullivan, Castillo, & Bellotti, 1991;Almeida & Vitorino, 1997;Vandenberg, 2002;Majka & Robinson, 2009;González & Gordon, 2009)

Associations Reported
A total of ten associations were recorded, six parasitoid-host and four predator-prey, three of which are new: parasitoids of the genus Eurytoma (Hymenoptera: Eurytomidae) and Hambletonia (Hymenoptera: Encyrtidae) associated with A. takahashii and S. sacchari, respectively; and the predator of the genus Diadiplosis (Diptera: Cecidomyiidae) with a S. sacchari.All associations registered during this work are new reports for the state of São Paulo.Anagyrus saccharicola and Mariola flava (Hymenoptera: Encyrtidae) are the first time in Brazil (Table 1).

Most Common Natural Enemies, and Other Comments
The most frequently collected natural enemies were parasitoids A. saccharicola and M. aclerdae, obtained in 10 and 8 sample points, respectively, three of common occurrence.Among the predators were drosofilids of the genus Rhinoleucophenga and the cocinellid Hyperaspis sp. in three and two sample points, respectively.The parasitoids emerged from scale insects collected in 16 sample points, while predators were obtained in only six.
The sampling point where the number of parasitoid species, four being one associated with S. sacchari and A. takahashii was located in a sugar mill in the municipality of Fernandópolis, in a sugarcane field made up of plants at harvest point.The point where the highest number of predators was obtained, two dipterans and two parasitoids, refers to an area with a small cane plantation, semi-shaded, located in a small farm in the municipality of São Carlos, SP.
Although the two scale insects species studied can occur together, infesting the same plant structure, under the nodes of sheaths, the species of natural enemies obtained associated with each of them was distinct: seven for A. takahashii and five for S. sacchari.Among the natural enemies listed for these scale insects in the world, a single species common to both was recorded, the parasitic wasp Astymachus japonicus Howard, 1898, in China and India (Appendix A).
From the 15 species of natural enemies reported in association with S. sacchari and A. takahashii in Brazil, 10 species were found in this survey, in the state of São Paulo (Appendix A and B).The three species previously mentioned were associated with A. takahashii: R. obesa by Lima (1935)  The most frequent parasitoids were A. saccharicola and M. aclerdae.Anagyrus saccharicola is probably from Indo-Malayan region Timberlake (1932) has been introduced as a biological control agent of S. sacchari in several producers countries of sugarcane, as Hawaii, Australia and Egypt (Swezey, 1934;Carver, Inkerman, & Ashbolt, 1987;Abd-Rabou, 2002).In Neotropical region this parasitoid was first successfully introduced and established in Barbados from the East African in 1970, reducing the pest population by over 90% (Alam, 1972).
Posteriorly it was also introduced in other Caribbean and South America countries.In South America this species was registered in Bolivia, Colombia, Guiana, Peru and Venezuela (Salazar, 1972;De Santis, 1979;Pruett & Colque, 1984;Noyes & Hayat, 1994;De Santis & Fidalgo, 1994).In Brazil, this insect was probably accidentally introduced together with the pseudococcid.Mucrencyrtus aclerdae, probably original of Neotropical region, has a restricted distribution.Collected in association with A. takahashii, on sugarcane, in Alagoas and in Espírito Santo (De Santis, 1972;Culik & Ventura, 2011), it has a distribution expanded to the Southeast Region.
The largest number of parasitoid species, observed from a single sampling point, emerged from A. takahashii, collected on plants of a sugarcane plant in the region of Fernandópolis.In this area, the population of Aclerdidae was predominant in relation to the mealybug and higher than in other areas, which probably favored the obtaining of the largest number of parasitoid species.
The largest number of predators, two dipterans, one associated with S. sacchari and one, A. takahashii, was observed in a small plantation of sugarcane, in a farm in São Carlos.Although the leaf sheath provides a protected environment for both the scale insect and its natural enemies, the fly larvae were probably even more favored because of the semi-shaded environment, which reduces the chances of desiccation.
The knowledge about predators and parasitoids of sugarcane scale insects population has a great importance, since for this crop biological control has already been used as a strategy to reduce some of the main pests such as Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae) with the parasite Cotesia flavipes Cameron, 1891 (Hymenoptera: Braconidae) and Mahanarva spp. with the entomopathogenic fungus Metarhizium anisopliae (Metschnikoff) (Parra, 2015).
× EGY (Herting, 1972;Noyes & Hayat, 1994;Abd-Rabou, 2000;2007 × MAS (Lim & Pan, 1976) Specimens "voucher" were deposited in the following institutions: the scale insects in the Reference Collection of Insects and Mites (CRIA) of the Plant Protection Department of FCAV/UNESP-Jaboticabal, SP; the parasitoids, in the "Oscar Monte" Entomophagous Insects Collection (IB-CBE) of the Biological Institute of Campinas, SP; the Coccinelidae, in the Entomological Collection Padre Jesus Santiago Moure (DZUP-Department of Zoology, Federal University of Paraná) in Curitiba, PR; in the Museum of Zoology of the University of São Paulo (MZUSP), SP and the drosophilids in the Reference Collection of Insects and Mites (CRIA) of the Plant Protection Department of FCAV/UNESP-Jaboticabal, SP.

Table 1 .
Natural enemies associated with Aclerda takahashii(Kuwana, 1932)and Saccharicoccus sacchari (Cockerell, 1895) collected in the state of São Paulo in the present study, frequent prey and hosts and references in the state of Rio de Janeiro; and, R. obesa, C. nigrescens and M. aclerdae by De Santis (1972) in the state of Alagoas.