Diversity of arthropods on Acacia mangium (Fabaceae) and production of this plant with dehydrated sewage sludge in degraded area

Sewage sludge is an organic matter-rich material with abundant fractions of nitrogen and other macro and micronutrients, essential for plant growth and development such as Acacia mangium Willd. (Fabales: Fabaceae) used in recovering actions of degraded areas. The objective of this study was to evaluate over 24 months the abundance and diversity of chewing and pollinator insects and arthropod predators on A. mangium plants and the mass production and soil coverage by this plant, fertilized with dehydrated sewage sludge, in a degraded area. The experimental design was in randomized blocks with two treatments (with and without dehydrated sewage sludge) and 24 replications. The number of leaves per branch and branches per plant, defoliation percentage by chewing insects, soil cover and abundance of chewing and pollinator insects and arthropod predators were higher on A. mangium plants fertilized with dehydrated sewage sludge. Nasutitermes sp. (Blattodea: Termitidae) and Trigona spinipes F. (Hymenoptera: Apidae) were the most observed insects on trunks and leaves, respectively, of A. mangium plants fertilized with dehydrated sewage sludge. The A. mangium fertilization increases the populations of different insect and spider groups on this plant.


Experimental design
Acacia mangium seedlings were produced from seeds of around 5-year old trees grown at the ICA/ UFMG campus. Seeds were dried, dormancy-broken and treated with recommended bactericides/ fungicides before sowing following standard protocol used for Acacia (= Vachellia Wight & Arn.) farnesiana (L.) Willd. in Brazil [35]. Seeds were sown in 8 × 12 cm plastic polybags (a seed per plastic polybag) in a nursery with its ruff covered using black shed net, with media mixing with 30% organic compost, 30% clay soil, 30% sand and 10% of reactive natural phosphate (160 g seedling −1 ) in March 2014. The organic compost consisted of three parts, by volume: two parts of debris gardening pruning (≤ 5 cm) and one part of tanned nelore cattle Bos taurus indicus L., 1758 (Artiodactyla: Bovidae) manure. The mixture clay soil and sand was treated by a heating process at 80°C for 15 min. The soil pH of the pits was corrected with dolomitic limestone (i.e. an anhydrous carbonate mineral composed of calcium magnesium carbonate), increasing the base saturation to 50% [36]. Fritted trace elements (FTE), gypsum, micronutrients, natural phosphate and potassium chloride were added according to the soil chemical analysis for the Minas Gerais State [37]. Thirty-centimeter tall A. mangium seedlings were planted in pits (40 × 40 × 40 cm) spaced 2 m between them, in six parallel lines on flat terrain, spaced 2 m between lines, with four plants with and four without fertilization with dehydrated sewage sludge per line, in September 2014. These seedlings were irrigated twice a week until the beginning of the rainy season using water from a nearby river from when no additional water was provided. The plants were pruned using a razor sterilized with a solution of sodium hydroxide + sodium hypochlorite, when their branches reached 5 cm long, eliminating the additional shoots (i.e. others different from the leader shoot) and branches up to one-third of crown height, leaving only the leader shoot and lateral branches up to two-thirds of the crown height. The pruned parts of each plant (branches and shoots) were left between their respective planting lines. The experimental design was completely randomized in blocks with two treatments (20 l of dehydrated sewage sludge per pit or no dehydrated sewage sludge) and 24 replications with one plant each. The 20 l of dehydrated sewage sludge was placed in a single dose per pit at 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 A. mangium experimental site. The ETE is operated by the Minas Gerais Sanitation Company S.A.--'Companhia de Saneamento de Minas Gerais S.A. (COPASA)' with capacity to treat 217 m 3 sewage sludge d −1 . The efficiency of the system in terms of removal of organic matter is higher than 90%. The sewage sludge goes through a solarization process in coarse sand tanks for three months in the ETE reducing the thermotolerant coliform bacteria to a level accepted by the National Council for the Environment--'Conselho Nacional do Meio Ambiente (CONAMA)' (Resolution N o 375) of the Ministry of the Environment--'Ministério do Meio Ambiente' of Brazil for use in agriculture, which is less than 10 3 most likely number g -1 of total solids. The main chemical and biological characteristics of the dehydrated sewage sludge of this company were pH-H 2 O = 4.40, N = 10.4 mg Kg -1 , P = 2.9 mg Kg -1 , K = 5.8 mg Kg -1 , Cd = 0.1 µg g -1 , Pb = 56.9 µg g -1 , Cr = 46.7 µg g -1 and faecal coliforms = 4.35 most likely number g -1 after analysis carried out in a laboratory [8].

Insects and spiders
Insects and spiders (no multiply counted) were counted by visual observation biweekly on the adaxial and abaxial surfaces of the leaves between 07.00 and 11.00 at the apical, middle and basal parts of the canopy in the northern, southern, eastern and western directions, totaling 12 leaves plant −1 evaluation −1 on the 48 A. mangium trees of six-month old for 24 months. Insects and spiders were not removed from plants during the evaluation, except those collected for identification. The total sample effort was 27 648 leaves covering the entire plant (vertical and horizontal axes), capturing as many insect and spider species as possible, especially the rarest. Insects and spiders present on the trunk (chest height) were collected, and insect defoliation was evaluated visually by the leaf area losses on a 0-100% scale with 5% increments for removed leaf area [38,39] for the 48 trees per evaluation. At least, three specimens per insect or spider species were captured per collection using aspirator, stored in glass flasks with 70% ethanol or mounted, separated into morphospecies and sent for identification.

Ecological indices
Averages were made by reducing the data to single trees. Ecological indices (diversity, individual abundance and species richness) were calculated for each identified species in the treatments (with or without dehydrated sewage sludge) per tree using the software BIODIVERSITY PROFESSIONAL, Version 2 (©1997 The Natural History Museum) [40]. The diversity was calculated using Hill's formula [41,42] and the species richness with Simpson indices [43,44]. The predator (insects and spiders) and prey ratio on A. mangium was calculated per tree.

Statistics
Data on leaves per branch, branches per plant, percentages of soil cover by litter, grass and herbaceous plants, predator per prey ratio and defoliation, diversity, abundance and richness of chewing, defoliator and pollinator insect species, and arthropod predators (see the electronic supplementary material) were submitted to the non-parametric statistical hypothesis, Wilcoxon signed-rank test ( p < 0.05) [45] using the statistical analysis program 'Sistema para Análises Estatísticas e Genéticas (SAEG)', version 9.1 [46] supplied by the 'Universidade Federal de Viçosa'.
The Spearman correlation matrix, among the most significant characteristics, was calculated. The matrices were submitted to correlation networks [47]. The edge thickness was controlled by applying a cut-of-value 0.26 (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 by R Core Team [48]. The correlation network procedure was performed using the package qgraph [47].

Leaves per branch, branches per plant, leaves per tree, percentages of defoliation and soil cover
Leaves per branch and branches per plant, percentages of defoliation by chewing insects and soil cover (litter, grasses and herbaceous plants) were higher for A. mangium plants fertilized with dehydrated sewage sludge than for those without fertilization, but no effect was observed on the predator per prey ratio (table 1). The increase in the number of leaves per tree reduced the predator per prey ratio (figure 1).

Biodiversity and richness indexes
The biodiversity and richness indexes for chewing and pollinator insects, and spiders only were similar for A. mangium plants fertilized or not with dehydrated sewage sludge. On the other hand, the abundance of chewing (greater than 10 times) and pollinator (greater than 2 times) insects, and total royalsocietypublishing.org/journal/rsos R. Soc. open sci. 7: 191196 predators (greater than 1.4 times) were higher on plants fertilized with dehydrated sewage sludge. Moreover, the treatments did not affect the spider ecological indices, but plants fertilized had more biodiversity of total predators (table 2). The increase in the number of leaves per tree increased the abundance and richness of chewing insects besides the predators on A. mangium plants. Higher species richness of chewing insects resulted in bigger predator abundances, including spiders (figure 1).

Arthropods
The termite Nasutitermes sp. (Blattodea: Termitidae) and the Neotropical stick grasshopper Cephalocoema sp. (Orthoptera: Proscopiidae) numbers were higher on A. mangium trunks fertilized with dehydrated sewage sludge and without fertilization, respectively, while the number of all other chewing insect species was similar ( p < 0.05) between treatments. The Orthoptera chewers, the large South American grasshopper Tropidacris collaris Stoll, 1813 (Romaleidae) and the katydid Tettigoniidae and the Coleoptera Lordops sp. (Curculionidae) and Stereoma anchoralis Lacordaire, 1848 (Chrysomelidae) stood out in relation to the other chewing insects owing to their greater abundance on A. mangium plants, with or without dehydrated sewage sludge fertilization (table 3). The increase in the number of leaves per tree increased that of Nasutitermes sp. (figure 1).
The stingless bee, Trigona spinipes F., 1793 (Hymenoptera: Apidae) numbers were higher on A. mangium plants fertilized with dehydrated sewage sludge while those of the European honeybee, Apis mellifera L., 1758 and the stingless bee, Tetragonisca angustula Latreille, 1811 (Hymenoptera: Apidae) were similar ( p < 0.05) between treatments, yet both had lower abundance than the first species (table 4).   The wasp Polybia sp. (Hymenoptera: Vespidae), the jumping spider Salticidae (Araneae) and the praying mantis Mantis religiosa (Linnaeus, 1758) (Mantodea: Mantidae) numbers were higher ( p < 0.05) on A. mangium plants with or without, respectively, dehydrated sewage sludge, while those of all other predators insect and spider species was similar ( p > 0.05) between treatments (table 4). The increase in the abundance of pollinators, Tettigoniidae and T. collaris individuals increased that of predators as well as the species richness of chewing insects resulted in higher numbers of the lynx spider Oxyopidae (Araneae) (figure 1).

Discussion
The highest abundance of chewing, pollinator and predator insects on A. mangium fertilized with dehydrated sewage sludge and soil covered by litter were owing to the better development of these plants (e.g. >leaves per tree), similar to that of the flooded gum Eucalyptus grandis W. Hill ex Maiden (Myrtales: Myrtaceae) [22] and their higher nitrogen levels in a dehydrated sewage sludge [8] obtained from the same ETE of the current study.
The litter cover in the crown projection area of A. mangium plants fertilized with dehydrated sewage sludge resulted from the higher number of leaves and branches produced by these plants compared to the non-fertilized ones, important for reducing laminar erosion and increasing soil fertility [49,50], confirming the first hypothesis in which fertilized plants are better in the recovery process of degraded areas. Dehydrated sewage sludge is rich in organic matter and macronutrients such as nitrogen and phosphorus, besides micronutrients such as copper and zinc, favouring tree growth and development [51,52]. The recovery of degraded areas is slow, but the use of A. mangium fertilized with dehydrated sewage sludge is promising, because of its fast growth and development, efficient fix atmospheric nitrogen gas fixation, potential to improve soil quality and widespread use [24]. This agrees with the positive impact of dehydrated sewage sludge in the development of the Brazilian pine, Araucaria angustifolia  Table 2. Diversity index (DI), species richness (SR) and abundance (abund.) of chewing and pollinator insects, total predators and spiders on Acacia mangium (Fabales: Fabaceae) per tree (mean ± SE) with or without dehydrated sewage sludge. protozoans) in these treatments was low and similar with soils without fertilization or with liming and chemical fertilization [8]. These treatments did not surpass the maximum limits of annual addition and the permissible maximum levels of heavy metal concentrations in the soils, but the concentrations of lead in Z. mays and V. unguiculata grains reached values above the limits permitted for agricultural products, regardless of the addition of sewage sludge in the soil [8]. The greater abundance of chewing insects and defoliation on A. mangium plants fertilized with dehydrated sewage sludge is probably owing to the greater number of leaves serving as a better food source and quality for insects. This confirms the second hypothesis that the diversity and abundance of herbivorous insects and their predators are usually higher and with higher increase of chewing insects than predators (e.g. >leaves ≤predator per prey ratio) on trees with higher leaf mass [25,53,54]. These trees function as a BGI, but with a higher chance of rare species extinction on those with lower leaf mass [25,28,55]. In addition, the quantity of free amino acids and proteins is superior in plants with higher nitrogen fertilization, favouring herbivorous insects [56]. Interactions between insects and Acacia species plants show the potential of this plant to increase the biodiversity and recover  [57][58][59][60]. The dehydrated sewage sludge as a biofertilizer improved macrofauna recovery, including the scarab beetles Scarabaeidae (Coleoptera) larvae and adults in degraded soils of the Cerrado (Brazilian savannah) type biome area [55].
The presence of Nasutitermes sp., as the most abundant insect on A. mangium plant trunks fertilized with dehydrated sewage sludge may be owing to the organic matter richness of this fertilizer [8,51] and the higher litter production by this plant (e.g. >leaves ≥ Nasutitermes sp.). This insect can damage living or dead trees and processed wood, including root systems, although they caused galleries in the trunks without damaging or causing plant death [61]. Damage by Lordops sp., S. anchoralis, T. collaris and Tettigoniidae on A. mangium leaves and their greater abundance compared to that of other chewing insects is worrying. Tropidacris collaris damaged the swamp she-oak, Casuarina glauca Sieber (Fagales: Casuarinaceae) and white leadtree, Leucaena leucocephala (Lam.) de Wit (Fabales: Fabaceae) [62,63]. Meroncidius intermedius Brunner Von Wattenwyl, 1895 (Orthoptera: Tettigonnidae) damaged grasses and banana Musa spp. fruits (Zingiberales: Musaceae) [64] and Lordops sp. defoliated the diesel tree, Copaifera langsdorffii Desf. (Fabales: Fabaceae) [65], but there is no reports of S. anchoralis damaging commercial plants.
The number of pollinating insects being two times higher on A. mangium plants fertilized with dehydrated sewage sludge is probably owing to their larger canopy size, higher number of flowers and supporting a greater insect numbers [25,54], including pollinators, confirming the third hypothesis: greater BGI greater pollinating insects. In addition, nitrogen fertilization via dehydrated sewage sludge may have increased the pollen and/or nectar production and quality (more amino acids and protein) in A. mangium flowers, increasing pollinator attractiveness as observed for the higher attraction of Nicotiana L. (Solanales: Solanaceae) species flowers with better quality of nectar sugars and amino acids by different groups of pollinators (e.g. bats (Chiroptera), hummingbirds (Trochilidae) or moths (Lepidoptera)) in Wuppertal, Germany [66] and floral pollens with better quality by bumblebees, Bombus Latreille, 1802 (Hymenoptera: Apidae) and honeybees, Aphis species in Newcastle, United Kingdon [67]. The greater T. spinipes pollinator abundance on A. mangium plants, especially on those fertilized with dehydrated sewage sludge may be of low importance, because it can reduce pollination as reported for Cucurbitaceae (Cucurbitales) owing to insufficient pollen transportation (small body size) and/or chasing other pollinators, such as A. mellifera and T. angustula, by flying in flocks and with aggressive behaviour [68]. In addition, T. spinipes damages shoots and plant growth regions by removing fibres to construct their nests, as reported on A. mangium and L. leucocephala, that also had their leaves and shoots damaged [63,69]. The greater abundance of predator insects and spiders, on A. mangium plants fertilized with dehydrated sewage sludge, is probably owing to the higher number of chewing and pollinator insects on the plants (larger trees), that is, these predators followed their prey [70], confirming the fourth hypothesis: greater BGI greater predators. In general, the number of predator insect and spider species did not differ between A. mangium plants with or without dehydrated sewage sludge fertilization, but the number of spider species (30% higher on fertilized plants) and the abundance of the predatory wasp Polybia sp. were higher on fertilized plants. Spider predators reduced insect damage, mainly from defoliators, such as spiders in many agroecosystems in the USA [71], wolf (Araneae: Lycosidae) and sheet weaver (Araneae: Linyphiidae) spiders in winter barley, Hordeum vulgare L. (Poales: Poaceae) fields situated in differently structured landscapes in Uppsala, Sweden [72], wandering spiders (Araneae: Ctenidae) in agroecosystems in Italy [73] and spiders in pequi, Caryocar brasiliense Cambess. (Malpighiales: Caryocaraceae) trees in Minas Gerais State, Brazil [26]. Predatory wasps (Vespidae) are important natural enemies in agricultural systems such as Brassica campestris L. and kale, Brassica oleracea L. var. acephala DC., Arabian coffee, Coffea arabica L. (Gentianales: Rubiaceae) and tomato, Solanum lycopersicon L. (Solanales: Solanaceae), preying mainly on caterpillars and leaf miners (Lepidoptera) in several regions of Brazil [74][75][76][77]. Sewage sludge increased the ground beetle Carabidae (Coleoptera) species richness in the area of Oxford, USA [78].

Conclusion
To summarize, the larger A. mangium crown (>BGI) fertilized with dehydrated sewage sludge increases soil cover (e.g. litter) and the abundance of chewing (>defoliation) and pollinator insects and arthropod predators, showing that this plant is adequate for recovering degraded areas using this fertilization. The presence of Nasutitermes sp. on A. mangium plant trunks fertilized with dehydrated sewage sludge may be owing to the organic matter richness of this fertilizer and the higher litter production by this plant (e.g. >leaves ≥ Nasutitermes sp.), but without damaging or causing plant death. On the other hand, Lordops sp., T. collaris and Tettigoniidae damaged leaves of A. mangium and this is worrying because these insects are pests in other economically important crops. The greater T. spinipes pollinator abundance on A. mangium plants is a problem owing to this insect damaged their shoots and plant growth regions.
Ethics. No specific permits are required to plant Acacia mangium in Brazil. The laboratory and field studies did not involve endangered or protected species.
Data accessibility. All data generated or analysed during this study are included in this manuscript.