Phytophagous insects and natural enemies on Sapindus saponaria L. (Sapindales: Sapindaceae) plants fertilized with or without dehydrated sewage sludge

Abstract Management programs and efficient techniques are necessary to recover degraded ecosystems. The sewage sludge is rich in nitrogen (N) and with the potential to fertilize Sapindus saponaria L. (Sapinales: Sapindaceae), used in the recovery of degraded areas; this can affect the insect fauna. The study's objective was to evaluate, for 24 months, the abundance of chewing insects, dipterans, pollinators, and predators on S. saponaria plants fertilized with or without dehydrated sewage sludge in a degraded area. The experimental design was completely randomized (with the same characteristics) with two treatments (with or without dehydrated sewage sludge) and 24 replicates, each with one plant. The abundance of Anastrepha sp. (Tephritidae), Cerotoma sp. (Chrysomelidae), Curculionidae, Musca domestica L. (Muscidae), Mantis religiosa L. (Mantodea: Mantidae), Oxyopidae, Salticidae, Tettigoniidae (Orthoptera), and Teudis sp. (Anyphaenidae) was higher on fertilized plants. The abundances of Teudis sp. and Tmarus sp. (Thomisidae) and M. religiosa and Teudis sp. were positively correlated with chewing insects and Diptera, respectively. The population increase of insects and spiders on S. saponaria plants fertilized with dehydrated sewage sludge (bigger crowns) has shown to be suitable for recovering degraded areas with a higher number of niches and better food quality, improving the ecological indices of the area.


Experimental design
Sapindus saponaria seedlings were produced from seeds of trees grown at the ICA/UFMG.Seedlings were planted in plastic bags (8 x 12 cm) in a nursery with a substrate mixed with 30% organic compost, 30% clay soil, 30% sand, and 10% reactive natural phosphate (160 g seedling -1 ) in March 2014.The organic compost consisted of three parts by volume: two parts of gardening pruning debris (≤ 5 cm) and one part of tanned bovine manure.The soil pH in the pits (40 × 40 × 40 cm) was corrected with dolomitic limestone, increasing the base saturation to 50% (Kopittke and Menzies, 2007).Natural phosphate, gypsum, fritted trace elements (FTE), potassium chloride, and micronutrients were added according to the soil analysis (Nouvellon et al., 2012).One 30 cm high S. saponaria seedling was planted per pit spaced 2 m between them, in six parallel lines on flat terrain (similar characteristics), spaced 2 m between lines, four plants with and four without fertilization with dehydrated sewage sludge/line, in September 2014.These seedlings were irrigated twice a week from the beginning of the rainy season until no additional water was provided.The plants were pruned with a razor sterilized per plant when their branches reached 5 cm long, eliminating the additional ones and those up to 1/3 of crown height, leaving only the best stem.The pruned parts of each plant were left between their respective planting lines.The experimental design was completely randomized with two treatments (20 L of dehydrated sewage sludge/pit or no dehydrated sewage sludge) and 24 replications with one plant each.Twenty liters of dehydrated sewage sludge were placed per pit in a single dose during planting.
Dehydrated sewage sludge (5% moisture content) was collected at the sewage treatment plant -"Estação de Tratamento de Esgoto (ETE)" in the municipality of Juramento, Minas Gerais State, Brazil, about 40 km from the S. saponaria experimental site.The ETE is operated by the Minas Gerais Sanitation Company -"Companhia de Saneamento de Minas Gerais S.A. (COPASA)" with the capacity to treat 217 m 3 sewage day -1 .This system removes more than 90% of the organic matter.The sewage sludge passes through a solarization process in coarse sand tanks during three months in the ETE, which reduces the thermotolerant coliforms to a level accepted by the National Council for the Environment -"Conselho Nacional do Meio Ambiente (CONAMA)" (Resolution Nº 498) of the Ministry compound saponin (Tsuzuki et al., 2007;Lorenzi, 2008).Insects associated with S. saponaria are poorly studied, but insecticidal properties (e.g., trypsin inhibitors) of leaf and fruit extracts of this plant were toxic to Trigona spinipes (Fabr.)(Hymenoptera: Apidae) (Macedo et al., 2011).
Sewage sludge, a residue rich in organic matter, has the potential for fertilization or the production of forest seedlings.This material is indicated for forest plantations and in the recovery of degraded areas to minimize the risk of toxic elements entering the human food chain (Kimberley et al., 2004;Martins et al., 2016), but its use can affect the fauna of insects (e.g., > N) (Jansson and Ekbom, 2002;Leite et al., 2011;Taiz et al., 2017).As a fertilizer, sewage sludge in agriculture and forestry can reduce production costs and environmental problems (Caldeira et al., 2014;Martins et al., 2016).
Insect diversity can be used to monitor the recovery of degraded areas as they respond to environmental changes through mutualistic relationships and pollination (Santos et al., 2006;Barah and Bones, 2015;Kishi et al., 2017).With large numbers of families and species, Coleoptera, Lepidoptera, and Hymenoptera (e.g., ants and bees) are indicators of the recovery of degraded areas (Barbieri Junior and Dias, 2012;Komonen et al., 2015;Kishi et al., 2017).Nutritional indices, chemical defenses, and factors, such as fertilization and age of host plants, impact the diversity of phytophagous insects and their natural enemies, including spiders (Bowers and Stamp, 1993;Coley and Barone, 1996;Leite et al., 2011).Sewage sludge increases plant development because it is rich in phosphorus and nitrogen -macroelements -and Cu and Zn -micronutrients, and consequently affects insects (Mass, 2010).
This study aimed to evaluate the abundance of chewing insects, dipterans, pollinators, and predators on S. saponaria plants and its defoliation by insects when fertilized with or without dehydrated sewage sludge, for 24 months, in a degraded area.The hypotheses tested were: i) fertilized plants will be larger (> BGI) with a higher abundance of phytophagous insects (Ferrier and Price, 2004;Espírito-Santo et al., 2007;Leite et al., 2017) and ii) the predators follow their prey (Auslander et al., 2003;Leite et al., 2017).

Insects and spiders
Insects and spiders were counted by visual observation biweekly on the leaf adaxial and abaxial surfaces between 7:00 and 11:00 A.M. at the apical, middle, and basal canopy parts in the northern, southern, eastern, and western directions, with 12 leaves/plant/evaluation on the six months old 48 S. saponaria saplings (young trees) during 24 months.Insects and spiders were not removed from the plants during the assessment.The total sample effort was 27,648 leaves covering the entire plant (vertical and horizontal axes) to capture as many insect and spider species as possible, including the rarest.Insects and spiders found on the trunk (chest height) were collected, and defoliation was evaluated visually by the leaf area losses on a 0-100% scale with 5% increments for the leaf area removed (Sastawa et al., 2004;Mizumachi et al., 2006) for the 48 saplings/evaluation.
The evaluator approached carefully, firstly assessing the adaxial leaf surface and, if it was impossible to visualize the abaxial one, with a delicate and slow movement, lifting the leaf to visualize it.Insects with greater mobility that flew on approach were counted as long as they were recognized (e.g., Order).During the evaluation, the arthropods (insects and spiders) were not removed from the saplings.A few arthropod specimens (up to 3 individuals) per species were collected using an aspirator (two hours per week) at the beginning of the study (between transplantation and first evaluation, six months after), stored in flasks with 70% alcohol, separated into morphospecies, and sent to specialists for identification.Any visible arthropod not yet computed in previous evaluations was collected, coded, and sent to a taxonomist of its group.

Ecological Indices
Each replication (saplings) is the individuals collected on 12 leaves (three heights and four sides of the saplings).The ecological indices of abundance were calculated per group, for example, pollinators and treatment (with or without dehydrated sewage sludge)/tree using the BioDiversity Professional© software, Version 2 (1997) (Krebs, 1989).Abundance was the total number of individuals (Begon et al., 2007) per tree.

Statistics
Data of percentage of defoliation, numbers of chewing, defoliator, Diptera, pollinator, spiders, and predator insect species were submitted to the non-parametric statistical hypothesis, Wilcoxon signed-rank test (P< 0.05) (Wilcoxon, 1945) using the statistical analysis program "Sistema para Análises Estatísticas e Genéticas (SAEG)," version 9.1 (UFV, 2007) (Supplier: "Universidade Federal de Viçosa").The data collected did not present a normal distribution.For this reason, they were analyzed using the non-parametric Wilcoxon test as the most powerful test locally among all the classification methods (Salov, 2014).Interactions between groups of phytophagous insects and their predators were analyzed by regression (P <0.05) with this statistical program.Simple equations (linear and quadratic responses) were selected based on the criteria: i) distribution of the data in the figures, ii) the parameters used in these regressions were the most significant ones (P <0.05), iii) P <0.05 and F of the analysis of variance of these regressions, and iv) the determination coefficient of these equations (R 2 ).The Spearman correlation matrix (linear response) was calculated among the most significant characteristics.
The matrices were submitted to correlation networks (Epskamp et al., 2012).The edge thickness was controlled by applying a cut of 0.26 value (from which the Spearman correlation became significant, meaning that only edges with |r ij |≥ 0.26 were highlighted).These analyses were performed using the R software version 3.4.1 (R Core Team, 2014).The correlation network procedure was performed using the package qgraph (Epskamp et al., 2012).(Blattodea: Termitidae), whose major visible damage was the formation of galleries on the plant trunk, was similar between treatments (Table 1).The number of leaves/saplings was positively correlated with those of Cerotoma sp., Curculionidae, and defoliation (Figure 1).

Discussion
The greater abundance of phytophagous arthropods Cerotoma sp., Curculionidae, and Tettigoniidae and defoliation in fertilized plants is due to the greater number of leaves/plant (>BGI), confirming the first hypothesis: >BGI> phytophagous insects (Ferrier and Price, 2004;Leite et al., 2017).Fertilization increases the size of trees and, consequently, the diversity and abundance of phytophagous insects (Ferrier and Price, 2004;Espírito-Santo et al., 2007;Leite et al., 2017) as they function, on a smaller scale, as a biogeographic island, where rare species are less likely to go extinct (Kitahara and Fujii, 1997;Burns, 2016;Leite et al., 2017).The damage by Cerotoma sp. and Curculionidae, defoliators, or boring pests of plants such as Casuarina glauca Sieber (Casuarinaceae), Leucaena leucocephala (Lam.)De Wit (Fabaceae), Glycine max (L.)Merrill (Fabaceae) and Zea mays L. (Gramineae) (Augustin et al., 2012;Lawal et al., 2014), on S. saponaria was low (≈5%, < control level = 20%).Despite the incidence of termites in both treatments, no damage was detected on the plants, similar to that on Acacia auriculiformis A. Cunn plants.ex Beth and Acacia mangium Willd.(Fabales: Fabaceae), fertilized with sewage sludge, where the increase in the number of leaves per tree favored the increase of Nasutitermes sp.(Dourado et al., 2020;Silva et al., 2020;Mota et al., 2021).This was Arthropods on Sapindus saponaria saplings attributed to the greater contribution of organic matter in the sewage sludge (Dourado et al., 2020;Silva et al., 2020;Mota et al., 2021) as well as higher litter production per plant (Dourado et al., 2020;Silva et al., 2020).Termites can damage living or dead trees, including root systems and processed wood (Albuquerque et al., 2014).Despite the construction of galleries, in this study, the termites did not damage the trunks or cause the death of plants.

Conclusions
The largest canopy of S. saponaria (>BGI) fertilized with dehydrated sewage sludge increased soil cover (e.g., litter) and the abundance of phytophagous insects and their predators on these plants, indicating that it is a promising method to recover degraded areas.The plants fertilized with dehydrated sewage sludge host a high number of chewing insects, pollinators, and predators, due to the greater number of niches and better food quality, improving the ecological indices of the area.

Table 2 .
Abundance of spiders and predators and pollinators insects on Sapindus saponaria saplings (mean ± SE) with or without dehydrated sewage sludge.
n = 24 per treatment; VT* = value of the test.