Article Dynamics of Aboveground Natural Enemies of , and after Application of Paranosema locustae in Rangeland

Wang-Peng Shi 1,2,* , Xiao-Yu Wang 2, Yue Yin 2, Yu-Xing Zhang 2, Um-e-Hani Rizvi 2, Shu-Qian Tan 2, Chuan Cao 2, Hong-Yan Yu 3 and Rong Ji 1,* 1 College of Life Science, International Cooperative Research Centre for Cross-border Pest Management in Central Asia, Xinjiang Normal University, Urumqi 830054, China 2 Department of Entomology, China Agricultural University, Beijing 100193, China 3 Qinghai Rangeland Work Station, Xining 810008, China * Correspondence: [email protected] (W.-P.S.); [email protected] (R.J.)

 Received: 3 June 2019; Accepted: 19 July 2019; Published: 26 July 2019 

Abstract: Substantial harm to ecosystems from the use of chemical pesticides has led to an increasing interest in the use of biopesticides to control grasshoppers in rangelands, including China. One such potential biopesticide for control of grasshoppers is the fungus Paranosema locustae. In this study, the dynamics of aboveground natural enemies of grasshoppers and diversity 0–9 years after application of P. locustae were investigated in rangeland in Qinghai Plateau, China. We found that the number of species and of individuals of aboveground natural enemies increased by 17–250% and 40–126%, respectively, after spraying P. locustae, and that the main natural enemies showed three peaks after treatment. The conventional indices of species diversity (H’) and evenness (J’) increased by 11–267% and 13–171%, respectively, after treatment with P. locustae. The results showed the positive effects of P. locustae on aboveground natural enemies and biodiversity in an arthropod community in Chinese rangeland. Paranosema locustae is thought to be a safe biological control agent for management in Northwestern China.

Keywords: Paranosema locustae; biodiversity; grasshoppers; biological control

1. Introduction More than 40 species of pest insects affect some or all of the 400 million hectares of grasslands in China. Of these species, grasshoppers are the most important, causing the largest losses in pastures and crops [1]. In an average year, more than 15–20 million hectares of grassland in northern China experience grasshopper outbreaks, and such outbreaks are common on the Qinghai–Tibet Plateau. These outbreaks have led to the need for continued research on biological control, because of the potential damage to the environment and rangeland ecosystems from the widespread use of chemical pesticides. The need for a biological alternative is especially important for the Qinghai–Tibet Plateau in China. Lots of entomopathogens have been evaluated for grasshopper control, including Paranosema locustae Canning (Microsporidia: Nosematidae), Metarhizium spp [2], Malamoeba locustae (King et Taylor) [3], and Beauveria bassiana (Balsamo) Vuillemin. Meanwhile, Metarhizium acridum (Driver & Milner) J.F. Bisch., Rehner & Humber (Green Muscle®, Green Guard®) is effective in controlling the desert locust and , Chortoicetes terminifera Walker [4]. More than 120 species of grasshoppers are susceptible to P. locustae [5]. The high spore production of P. locustae- infected hosts, the vertical transmission of the disease through its host, and its wide host range within acridid grasshoppers gives it the potential for wide application for controlling grasshoppers in grasslands.

Insects 2019, 10, 224; doi:10.3390/insects10080224 www.mdpi.com/journal/insects Insects 2019, 10, 224 2 of 9

Over the past twenty years, more than 1 million hectares have been treated with P. locustae in China, demonstrating the efficacy of this biological agent for managing grasshoppers [6]. P. locustae remains in grasshopper populations even in high ultraviolet rays’ area Qinghai Plateau for many years, and the density of primary grasshopper species was kept below EIL in these treated areas [7]. Predatory natural enemies are important vectors for this microsporidian disease [8]. A great deal of attention has been paid to the effects of the pathogen on non-target , especially on the main natural enemies of grasshoppers and biodiversity, in general, in rangeland. The current study examines the effects of P. locustae on the principal groups of aboveground natural enemies of grasshoppers and arthropod biodiversity in a rangeland arthropod community over a nine-year period following application in the Qinghai–Tibet Plateau.

2. Materials and Methods

2.1. Entomopathogen Formulations P. locustae from North America was propagated at the China Agricultural University, Beijing, China, by inoculating laboratory-reared third instar Locusta migratoria with spores via oral inoculation in water at 1.0 105 spores/mL. After rearing inoculated grasshoppers for 30–40 days, spores were × collected from cadavers, with an average yield of 2.0 1010 spores per locust. The spores were × formulated in water (1.0 1010 spores/mL water) and stored in a freezer [8]. When most grasshoppers × in the field were in the third instar, formulated spores were applied as an ultra-low-volume spray using a Taishan 1806 mobile fogging machine (Taian Pesticide Machine Ltd., Taian, China). The formulated dose level was 7.5 109 P. locustae spores in 12 L water/hm2. × 2.2. Experimental Plots The experimental sites covered an area of 15,000 hectares in Tianjun County (Altitude, 3417 m; 99◦020E; 37◦180N), Qinghai–Tibet Rangeland. There were three plots for both treated and control; every plot was 1500 hectares of natural pasture; and there were discrete areas separated by untreated terrain.

2.3. Sampling For nine years after treatment with P. locustae, grasshoppers and aboveground main natural enemies [9], such as formicid ants, the carabid beetle Aristochroa venusta Tschitschérine, the scelionid wasp Scelio nikolski, the meloid Epicauta gornami Marseul, the sarcophagid fly Sarcophaga filipievi (Rodhain), the bombyliid fly Systoechus sp., spiders, and the agamid lizard Phrynocephalus vlangalii Strauch were collected from the treated and control area at the end of August every year; the initial time of 0 is the finding for the year of P. locustae application. The different stage of natural enemies and arthropods above described were collected with an insect suction collector (UNIVAC, Burkard Agronomics, England) at all treated and untreated plots [10], and insect densities were estimated within square aluminium frames (length width height: 1 1 0.5 m), making 50 such collections × × × × to estimate insect density within each plot. All insects were collected from the areas defined by the aluminium frames with suction provided by the D-vacs, and all collected material was placed individually by sample into plastic bags before being stored in a freezer for later identification of insect contents. For common species, including Oedaleus decorus asiaticus Bey-Bienko, Dasyhippus barbipes (F.-W.), Bryodema luctuosum luctuosum Stoll, Angaracris barabensis Pallas, and Myrmeleotettix palpalis Zubovski besides the natural enemies above described, the abundance (number of individuals) was noted and all specimens collected were identified.

2.4. Statistical Analysis The conventional indices of species diversity (Shannon diversity, H’) and evenness (J’ = H’/ln (S’) [11] were used to compare the species richness and species diversity of arthropod communities of the Qinghai–Tibet Plateau region in the study area. The indices were calculated using data from a InsectsInsects2019 2019, 10, ,10 224, x FOR PEER REVIEW 3 of 310 of 9

90 the Qinghai-Tibet Plateau region in the study area. The indices were calculated using data from a 91 seriesseries of of sample sample collections collections made made in in the the study study area.area. The variance variance of of the the Shannon Shannon index index and and evenness evenness 92 waswas calculated calculated as as per per [12 [12],13 [13].]. 93 One-wayOne-way ANOVAs ANOVAs were were used used toto comparecompare thethe densitydensity and and rate rate of of infection infection in in grasshoppers grasshoppers 94 betweenbetween treated treated and and untreated untreated areas areas with with least-significant least-significant difference difference (LSD) (LSD) analysis analysis used forused multiple for 95 comparisonsmultiple comparisons to measure to themeasure difference the difference between between different different treatments. treatments. SPSS 17.0 SPSS was 17.0 used was forused the 96 calculationsfor the calculations and analysis. and analysis.

97 3. Results3. Results

98 3.1.3.1. Population Population Fluctuations fluctuations ofof Grasshopper’sgrasshopper’s natural Natural enemies Enemies 99 TheThe sarcophagid sarcophagid fly S. filipievi S. filipieviis one is ofone the of main the parasitoid main parasitoid of grasshoppers, of grasshoppers, and three and population three 100 peakspopulation were observed peaks were for observed this species, for this in the spec firsties, (2.35in the first0.56 (2.35 individuals ± 0.56 individuals/m/m2), fifth (10.902), fifth (10.901.48/m ± 2), 2 2 ± ± 101 and1.48/m eighth) and (3.73 eighth1.07 (3.73/m2) ± year 1.07/m after) year treatment after treatment with P. locustae with P., andlocustae the, sumand the of all sum counts of all of countsS. filipievi of ± 102 showedS. filipievi significant showed di significantfference for difference treatment for versus treatment control vs ( Fcontrol= 17.373, (F =df 17.373,= 8, 18, dfp =< 8,0.05), 18, p but<0.05),S. nikolski but 103 (F =Scelio2.21, nikolskidf = 8, (F 18, = 2.21,p > 0.05) df = and8, 18,Systoechus p >0.05) and(F =Systoechus1.454, df (=F 8,= 1.454, 18, p >df0.05) = 8, 18, showed p >0.05) any showed insignificant any 104 insignificant differences between treatment and control respectively (Fig. 1). differences between treatment and control, respectively (Figure1).

14 Sarcophaga filipievi(PL) Sarcophaga filipievi(CK) A 12 2 10 8 6 Individuals Individuals /m 4 2 0 -2 012345789 Years after treatment with Paranosema locustae 105

Scelio nikolski(PL) 2.5 B Scelio nikolski(CK) 2 2 1.5

1

Individuals Individuals /m 0.5

0 012345789 -0.5

-1 Years after treatment with Paranosema locustae 106

Figure 1. Cont. InsectsInsects2019 2019, 10, ,10 224, x FOR PEER REVIEW 4 of 410 of 9

0.6 Systoechus(PL) C Systoechus(CK) 2 0.4

0.2 Individuals Individuals /m

0 012345789

-0.2 Years after treatment with Paranosema locustae 107 Figure 1. Densities of grasshopper parasitoids in study area, 0–9 years after treatment with Paranosema locustae. 108 Figure 1. Densities of grasshopper parasitoids in study area, 0-9 years after treatment with 109 AnalysisParanosema (one-way locustae. ANOVA) of differences of the sum of all counts of parasitoids of grasshoppers between the control (CK) and treated area (PL)—A: S. filipievi (F = 17.373, df = 8, 18, p < 0.05); B: 110 S. nikolskiAnalysis,(F = 2.21, (one-waydf = 8, 18,ANOVA)p > 0.05); of C:differencesSystoechus of,( Fthe= 1.454, sum dfof= all8, 18,countsp > 0.05). of parasitoids of 111 grasshoppersA predator, between the carabid the control beetle (CK)A. venusta, and treatedalso showedarea (PL). three A: Sarcophaga peaks, in filipievi the first (F (0.55= 17.373,0.16 df =/ m8, 2), 112 18, p <0.05); B: Scelio nikolski, (F = 2.21, df = 8, 18, p >0.05); C: Systoechus, (F = 1.454, df = 8, 18, p± >0.05). fifth (1.64 1.03/m2), and ninth (0.53 0.10/m2) year after treatment. Ants (Formicidae) also showed 113 A predator,± the carabid beetle A.± venusta, also showed three peaks, in the first (0.55 ± 0.16/m2), three peaks, in the third (2.77 0.31/m2), seventh (3.03 1.12/m2), and ninth (2.35 0.61/m2) year after 114 Insectsfifth 2019 (1.64, 10, ±x 1.03/m2), and ninth± (0.53 ± 0.10/m2) year after± treatment. Ants (Formicidae)± also showed5 of 11 treatment, but the sum of all counts of Formicidae (F = 1.321, df = 8, 18, p > 0.05), A. venusta (F = 0.908, 115 three peaks, in the third (2.77 ± 0.31/m2), seventh (3.03 ± 1.12/m2), and ninth (2.35 ± 0.61/m2) year after df = 8, 18, p > 0.05), E. gornami (F = 1.19, df = 8, 18, p > 0.05), Araneae (F = 2.158, df = 8, 18, p > 0.05),2 116 treatment,A predator, but thethe sum carabid of all beetle counts A. of venusta, Formicidae also F showed= 1.321, df three = 8, 18, peaks, p >0.05), in the A. venustafirst (0.55 (F = ± 0.908, 0.16/m df ), P. vlangalii F 2 df p 2 117 fifthand= 8,(1.64 18, p± >0.05),1.03/m( =E.), 0.991, gornamiand ninth (=F =8,(0.53 1.19, 18, ± df >0.10/m =0.05) 8, 18,) showed pyear >0.05), after noAraneae treatment. significant (F = 2.158,Ants difference (Formicidae)df = 8, for 18, treatmentp >0.05) also andshowed versus P. 118 threecontrol,vlangalii peaks, respectively (F in = the 0.991, third (Figure df (2.77= 8,2 ).18,± 0.31/m p >0.05)2), seventh showed (3.03 no significant ± 1.12/m2), diff anderence ninth for (2.35 treatment ± 0.61/m vs2) yearcontrol after 119 treatment,respectivelyAnalysis but (one-way (Fig.the sum2). ANOVA)of all counts of di offf erencesFormicidae of the (F sum = 1.321, of all df counts = 8, 18, of predatorsp > 0.05), ofA. grasshoppersvenusta (F = 0.908,between df = the 8, 18, control p > 0.05), (CK) E. and gornami treated (F area = 1.19, (PL)—A: df = 8, Formicidae18, p > 0.05), ( FAraneae= 1.321, (Fdf = =2.158,8, 18, dfp => 8,0.05); 18, p B:> 0.05),Aristochroa and P. venusta vlangalii(F = (0.908,F = 0.991,df = df8, 18,= 8,p 18,> 0.05);p > 0.05) C: Epicauta showed gornami no significant(F = 1.19, differencedf = 8, 18, forp >treatment0.05); D: versusAraneae control, (F = 2.158, respectivelydf = 8, 18, (Figurep > 0.05); 2). E: Phrynocephalus vlangalii (F = 0.991, df = 8, 18, p > 0.05).

120

Figure 2. Cont. 2 B Aristochroa venusta(PL) Aristochroa venusta(CK) 1.5

1

0.5 Individuals Individuals

0 0246810 -0.5

-1 Years after treatment with Paranosema locustae

Insects 2019, 10, x 5 of 11

A predator, the carabid beetle A. venusta, also showed three peaks, in the first (0.55 ± 0.16/m2), fifth (1.64 ± 1.03/m2), and ninth (0.53 ± 0.10/m2) year after treatment. Ants (Formicidae) also showed three peaks, in the third (2.77 ± 0.31/m2), seventh (3.03 ± 1.12/m2), and ninth (2.35 ± 0.61/m2) year after treatment, but the sum of all counts of Formicidae (F = 1.321, df = 8, 18, p > 0.05), A. venusta (F = 0.908, df = 8, 18, p > 0.05), E. gornami (F = 1.19, df = 8, 18, p > 0.05), Araneae (F = 2.158, df = 8, 18, p > 0.05), and P. vlangalii (F = 0.991, df = 8, 18, p > 0.05) showed no significant difference for treatment versus control, respectively (Figure 2).

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2 B Aristochroa venusta(PL) Aristochroa venusta(CK) 1.5

1

0.5 Individuals Individuals

0 0246810 -0.5 Insects 2019, 10, x 6 of 11 -1 Years after treatment with Paranosema locustae

0.6 Epicauta gornami(PL) Epicauta gornami(CK) C 0.5 2 0.4 0.3 0.2

Individuals /m Individuals 0.1 0 -0.1 0246810

-0.2 -0.3 Years after treatment with Paranosema locustae

2 D Araneae(PL) Araneae(CK)

2 1.5

1

Individuals /m Individuals 0.5

0 0246810 -0.5 Years after treatment with Paranosema locustae Figure 2. Cont. E 0.8 Phrynocephalus vlangalii(PL) Phrynocephalus vlangalii(CK)

2 0.7 0.6 0.5 0.4

Individuals /m Individuals 0.3 0.2 0.1 0 -0.1 0246810 Years after treatment with Paranosema locustae

Figure 2. Densities of grasshopper predators 0–9 years after treatment with P. locustae.

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0.6 Epicauta gornami(PL) Epicauta gornami(CK) C 0.5 2 0.4 0.3 0.2

Individuals /m Individuals 0.1 0 -0.1 0246810 -0.2 -0.3 Years after treatment with Paranosema locustae

2 D Araneae(PL) Araneae(CK)

2 1.5

1

Individuals /m Individuals 0.5

0 0246810 -0.5 Insects 2019, 10, 224 6 of 9 Years after treatment with Paranosema locustae

E 0.8 Phrynocephalus vlangalii(PL) Phrynocephalus vlangalii(CK)

2 0.7 0.6 0.5 0.4

Individuals /m Individuals 0.3

Insects 2019, 10, 0.2x 7 of 10 0.1 Analysis (one-way ANOVA) of differences of the sum of all counts of predators of grasshoppers between0 the control (CK) and treated area (PL)—A: Formicidae (F = 1.321, df = 8, 18, p > 0.05); B: Aristochroa-0.1 0246810 venusta (F = 0.908, df = 8, 18, p > 0.05); C: Epicauta gornami (F = 1.19, df = 8, 18, p > 0.05); D: Araneae (F = 2.158,Years df = after 8, 18, treatmentp > 0.05); E: withPhrynocephalus Paranosema vlangalii locusta (F = 0.991,e df = 8, 18, p > 0.05). Figure 2. Densities of grasshopper predators 0–9 years after treatment with P. locustae. Figure 2. Densities of grasshopper predators 0–9 years after treatment with P. locustae. 3.2.3.2. Diversity Diversity of ofA Arthropodsrthropods after after A Applicationpplication of of P. P. locustae locustae

NineNine years years after after treatment treatment with with P.P. locustae locustae, the, the total total number number of of species species and and individuals individuals of of all all the the namednamed species species collected collected (see (see above above described described)) increased increased by by 250% 250% and and 126% 126%,, respectively respectively,, in in areas areas treatedtreated with with P.P. locustae locustae. . At At the the same same time, time, the the conventional conventional indices indices of of species species diversity diversity (H') (H’) and and evennessevenness (J') (J’) increased increased by by 267% 267% and and 171%, 171%, respectively. respectively. Analysis Analysis of of overall overall data data showed showed that that there there werewere significant significant differences differences between between in in the the control control (CK) (CK) and and treated treated area area (PL (PL)) for for both both the the Shannon Shannon indexindex for for diversity diversity (H’) (H’) and and evenness evenness (J’) (J’) (F ( F= =14.647,14.647, df df= 9,= 50,9, 50,p < p0.05)< 0.05) and and evenness evenness (J’) (J’)(F = ( F7.354,= 7.354, df = df9, =50,9, p 50,< 0.05)p < 0.05) (Figure (Figure 3). 3).

Figure 3. Arthropod richness and evenness indices, 0–9 years after treatment in plots treated with P. locustae.

Figure 3. Arthropod richness and evenness indices, 0–9 years after treatment in plots treated with P. locustae.

Analysis (one-way ANOVA) of overall differences of diversity indices of arthropod between the control (CK) and treated area (PL): the Shannon index for diversity (H’) (F = 14.647, df = 9, 50, p < 0.05) and evenness (J’) (F = 7.354, df = 9, 50, p < 0.05).

3.3. Fluctuations of Grasshoppers among Years 0–9 Post-Treatment

At the time of treatment, grasshoppers were 15.76 ± 2.67 individuals/m2, but it declined by 98.9% by the fifth year after treatment with P. locustae. Grasshoppers increased after the sixth year and peaked to 13.56 ± 1.64 in year nine (Figure 4). Analysis (one-way ANOVA) of differences of the sum of all counts of grasshoppers between the control (CK) and treated area (PL) (F = 11.221, df = 9, 20, p < 0.05) was done.

Insects 2019, 10, 224 7 of 9

Analysis (one-way ANOVA) of overall differences of diversity indices of arthropod between the control (CK) and treated area (PL): the Shannon index for diversity (H’) (F = 14.647, df = 9, 50, p < 0.05) and evenness (J’) (F = 7.354, df = 9, 50, p < 0.05).

3.3. Fluctuations of Grasshoppers among Years 0–9 Post-Treatment At the time of treatment, grasshoppers were 15.76 2.67 individuals/m2, but it declined by 98.9% ± by the fifth year after treatment with P. locustae. Grasshoppers increased after the sixth year and peaked to 13.56 1.64 in year nine (Figure4). Analysis (one-way ANOVA) of di fferences of the sum of all ± counts of grasshoppers between the control (CK) and treated area (PL) (F = 11.221, df = 9, 20, p < 0.05) Insectswas 2019 done., 10, x 8 of 10

Figure 4. Mean densities ± SE (vertical bars) of grasshoppers in the study area, 0–9 years after Figure 4. Mean densities SE (vertical bars) of grasshoppers in the study area, 0–9 years after treatment treatment with P. locustae. ± with P. locustae.

AnalysisAnalysis (one (one-way-way ANOVA) ANOVA) of of differences differences of of the the sum sum of of all all counts counts ofof grasshoppers grasshoppers between between the the controlcontrol (CK) (CK) and and treated treated area area (PL (PL)) (F ( F= =1111.221,.221, df df= 9,= 20,9,20, p

4. 4.Discussion Discussion P. P.locustae locustae hashas been been found found to tobe be a useful a useful tool tool of ofgrasshopper grasshopper management management in inthe the rangeland rangeland [1,6] [1,.6 ]. AlthoughAlthough P.P. locustae locustae oftenoften causes causes only only low low-to-moderate-to-moderate levels ofof directdirect mortality mortality post-treatment post-treatment [14 [1],4] the, thecumulative cumulative usefulness usefulness of P. of locustaeP. locustaecan becan far be greater far greater [6,15 ].[6,1 In5 the]. I presentn the present study, westudy, found we increases found increasesin both the in both number the of number species of and species the number and the of individuals number of ofindividuals natural enemies of natural of grasshoppers enemies of in grasshoppersthe study area in over the the study nine-year area over period the after nine the-year application period ofafterP. locustae the application, demonstrating of P. an locustae indirect, demonstratingbenefit for controlling an indirect grasshoppers benefit for c withontrolling this bio-agent. grasshoppers with this bio-agent. InIn addition addition to to the the level level of of grasshopper grasshopper mortality mortality obtained obtained us usinging products products like like P. P. locustae locustae, , grasshoppergrasshopper management management programs programs should should consider consider the the sustainability sustainability of their of practices their practices and long-term and longeffects-term on effects the health on the of heal theth rangeland of the rangelan ecosystemd ecosystem [1]. A similar [1]. A impact similar of impactP. locustae of P.on locusta a rangelande on a rangelandsystem was system observed was observed in Xinjiang in Xinjiang and Inner and Mongolia Inner Mongolia provinces provinces and in and Argentina in Argentina [15,16 ],[1 but5,16], the butcauses the causes of the of long-term the long- etermffects effects post-treatment post-treatment with P. with locustae P. locustarequiree require further further research. research. InIn plots plots treated treated with with chemical chemical pesticides pesticides in inTianjun, Tianjun, the the carabid carabid beetle beetle A.A. venusta venusta andand the the lizard lizard P.P. vlangalii vlangalii numbers declined declined by by about about 70% 70% and 35%, and respectively, 35%, respectively, one year one after year treatment, after treatment,compared to comparedareas treated to areas with treatedP. locustae with, and P. locustae the chemically, and the treated chemically plots treated had to beplots re-treated had to be with re- pesticidestreated with after pesticides3–4 years after [1]. An3–4 importantyears [1]. concernAn important for the concern sustainable for the use sustainable of biological use pesticides of biological over wide pesticides areas is over wide areas is their effect on non-target native arthropods, especially natural enemies of the targeted pest. Arthropods are the most diverse group of organisms in ecosystems and include many species with biodiversity or pest control value. Here, we used diversity assessments of the natural enemy assemblages in P. locustae-treated areas to evaluate the impact of P. locustae on the main natural enemies and diversity in the study region. Although ants are highly diversified and exhibit several alimentary behaviors, most species of ants are to a greater or lesser degree predators or scavengers, and these ants we investigated like to feed on dead, dying, sick or molting grasshoppers in the grassland; so they were considered as a kind of natural enemy of grasshoppers in the experiment. We found that the common grasshopper natural enemies (Ants, A. venusta, S. nikolski, and S. filipievi) in the area were abundant, and that the conventional indices of species diversity (H') and evenness (J') showed significant increases, suggesting that P. locustae can be considered a safe biological agent to non-target arthropods, and its use advocated in grasshopper management. Another one, the infected grasshopper was easy to be predated by its predators because of slow

Insects 2019, 10, 224 8 of 9 their effect on non-target native arthropods, especially natural enemies of the targeted pest. Arthropods are the most diverse group of organisms in ecosystems and include many species with biodiversity or pest control value. Here, we used diversity assessments of the natural enemy assemblages in P. locustae-treated areas to evaluate the impact of P. locustae on the main natural enemies and diversity in the study region. Although ants are highly diversified and exhibit several alimentary behaviors, most species of ants are to a greater or lesser degree predators or scavengers, and these ants we investigated like to feed on dead, dying, sick or molting grasshoppers in the grassland; so they were considered as a kind of natural enemy of grasshoppers in the experiment. We found that the common grasshopper natural enemies (Ants, A. venusta, S. nikolski, and S. filipievi) in the area were abundant, and that the conventional indices of species diversity (H’) and evenness (J’) showed significant increases, suggesting that P. locustae can be considered a safe biological agent to non-target arthropods, and its use advocated in grasshopper management. Another one, the infected grasshopper was easy to be predated by its predators because of slow behavior, which is beneficial for natural enemies of grasshoppers. The predatory natural enemies of grasshoppers are important vectors for P. locustae, so the increases of natural enemies of grasshoppers improve the spread of the pathogen in the field. The density of Araneae has suffered a fall over the nine-year period, and we think that the rainfall and humidity declines have been detrimental to spider in recent years. The impact of Paranosema to native entomopathogeny of grasshopper will be researched further in the future. The sustained control effect of P. locustae on grasshoppers was demonstrated here; the increases of natural enemies of grasshoppers perhaps are a potential factor; P. locustae has been found persisting in grasshopper populations many years after application in rangeland [16]; and the persistence of the pathogen also is helpful for long-term control of grasshopper, because the common grasshoppers are susceptible to P. locustae collected in the grassland [7].

5. Conclusion The number of species and individuals of aboveground main natural enemies of grasshoppers and biodiversity of arthropods increased in regions treated with P. locustae. Preventive control of grasshoppers with biological pesticides P. locustae has been beneficial for keeping biodiversity of rangeland system.

Author Contributions: W.-P.S., X.-Y.W., H.-Y.Y., R.J. and C.C. conceived the study, developed the methodology, and contributed to the writing of the manuscript. X.-Y.W., Y.Y., Y.-X.Z., U.-e.-H.R., and S.-Q.T. conducted experiments and collected the data. W.-P.S. and H.-Y.Y. analyzed the data and conducted statistical analyses. W.-P.S. secured the funding for the research. All authors have read and approved the final article for submission. Funding: This research was funded by the National Key Program of China (2017YFD0201200), by the National Natural Science Foundation of China (31772221). Acknowledgments: English editing of this manuscript by Van Driesche Scientific Editing. Conflicts of Interest: The authors declare no conflict of interest for this publication. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References

1. Zhu, E.L. Sustainable management of locust in China. In National Extension Centre of Agricultural Technology; Agriculture Pres: Beijing, China, 1999; pp. 1–37. 2. Zimmermann, G.; Zelasny, B.R.; Kleespies, M.W. Biological control of African locusts by entomopathogenic micoorganisms. In New Trends in Locust Control; Wilps, H., Krall, S., Eds.; Deutsche Gesellschaft für TechnischeZusammenarbeit: Bonn, Germany, 1994; pp. 127–138. 3. Wilps, H.; Nasseh, O. Field tests with botanicals, mycopesticides, and chitin synthesis inhibitors. In New Trends in Locust Control; Wilps, H., Krall, S., Eds.; Deutsche Gesellschaft für TechnischeZusammenarbeit: Bonn, Germany, 1994; pp. 51–79. Insects 2019, 10, 224 9 of 9

4. Lomer, C.J.; Bateman, R.P.; Johnson, D.L.; Langewald, J.; Thomas, M. Biological control of locusts and grasshoppers. Ann. Rev. Entomol. 2001, 46, 667–702. [CrossRef][PubMed] 5. Lange, C.E. The host and geographical range of the grasshopper pathogen Paranosema (Nosema) locustae revisited. J. Res. 2005, 14, 137–141. [CrossRef] 6. Zhang, Z.H.; Yan, Y.H.; Zhang, Z.R.; Zheng, S.Y.; Xiang, S.Y.; Li, F.; Li, Y.S. The protection effect and significance on grassland ecosystem biodiversity of using Nosema locustae to control locusts. Pratacultural Sci. 2003, 9, 17–23. 7. Zhang, K.Q.; Xing, X.J.; Hou, X.M.; Tan, S.Q.; Chen, H.X.; Liu, P.P.; Shi, W.P.Population dynamics and infection prevalence of grasshopper (Orthoptera: ) after application of Paranosema locustae (Microsporidia). Egypt. J. Biol. Pest Control. 2015, 25, 33–38. 8. Shi, W.P.; Zheng, X.; Jia, W.T.; Li, A.M.; Camara, I.; Chen, H.X.; Tan, S.Q.; Liu, Y.Q.; Ji, R. Horizontal transmission of Paranosema locustae (Microsporidia) in grasshopper populations via predatory natural enemies. Pest Manag. Sci. 2018, 74, 2589–2593. 9. Ren, C.; Jing, X.; Shi, W. Effect of Nosema locustae for controlling grasshopper on natural enemy in the grassland. Heilongjiang J. Anim. Sci. Vet. Med. 2004, 2, 11–13. 10. Shi, W.P.; Chen, X.; Lu, F.; Guo, C. Persistence of Paranosema (Nosema) locustae (Microsporidia: Nosematidae) among grasshopper (Orthoptera: Acrididae) populations in the Inner Mongolia Rangeland, China. Biocontrol 2009, 54, 77–84. [CrossRef] 11. Pielou, E.C. The measurement of diversity in different types of biological collections. J. Theor. Biol. 1966, 13, 131–144. [CrossRef] 12. Pielou, E.C. Shannon’s formula as a measure of specific diversity: Its use and misuse. Am. Natur. 1966, 100, 463–465. [CrossRef] 13. Hurlbert, S.H. The nonconcept of species diversity: A critique and alternative parameters. Ecology 1971, 52, 577–586. [CrossRef][PubMed] 14. Lockwood, J.A.; Bomar, C.R.; Ewen, A.B. The history of biological control with Nosema locustae: Lessons for locust management. Entomol. Exp. Appl. 1999, 19, 333–350. [CrossRef] 15. Shi, W.P.; Peter, G.N.N. The disruption of aggregation behaviour of Oriental Migratory Locusts (Locusta migratoria manilensis) infected with Nosema locustae. J. Appl. Entomol. 2004, 128, 414–418. [CrossRef] 16. Lange, C.E.; Azzaro, F.G. New case of long-term persistence of Paranosema locustae (Microsporidia) in melanopline grasshoppers (Orthoptera: Acrididae: Melanoplinae) of Argentina. J. Inverteb. Pathol. 2008, 99, 357–359. [CrossRef][PubMed]

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