Sub-Saharan Africa for 2017 [29] (mapSPAM) were obtained from the International Food Policy Research Institute.

## 2.2. *Analysis*

We extracted Locust Hub reports for three regions: East Africa, the Middle East and the Eastern Region. East Africa region included Ethiopia (ET), Kenya (KE), Somalia (SO), Eritrea (ER), Djibouti (DJ), Uganda (UG), Tanzania (TZ), Sudan (SD&SU), South Sudan (SS), Egypt (EG) and D.R. Congo (CG). The country codes correspond to the "COUNTRYID" column in the Locust Hub database.

The Middle East region included Oman (OM), Saudi Arabia (SA), United Arab Emirates (AE&MU&TC), Yemen (YM&YE), Iraq (IQ), Jordan (JO), Bahrein (BH), Israel (IS), Kuwait) (KU&KW), Lebanon (LE) and Syria (SY).

The Eastern Region included India (IN), Pakistan (PK& PA), Iran (IR), and Nepal (NP). Our classification for the Eastern Region is based on observations that during recessions and even during upsurges, desert locust circulate between Iran, Pakistan and India. Only in a few situations do locusts invade the Eastern Region countries from outside, e.g., from Saudi Arabia to Iran, from Yemen/Somalia to India/Pakistan. This was also true during the current upsurge.

For each control report we extracted the country where it was applied and the type of control used. To align control data with 'Swarm' and 'Band' datasets from [24], we searched for reports that were reported within a 1 km radius and up to two days before the control operation. There remained a significant number of control reports within the criteria that did not correspond with the normal targets for control of 'Swarm' or 'Band' but with observations of the solitarious classifications, 'Adult' or 'Band'. It is likely that the observations of solitarious desert locusts may be inflated by misclassification of the remnants of former swarms and bands and by multiple reporting of the same insects, a common problem of crowd-sourced reporting of data. Accordingly control operations that were not matched to 'Swarms' and 'Bands' were assigned to a 'Not Assessed' ('NA') category.

Land cover type corresponding to the reported location of the control operation was extracted from CLC100 [25].

To align control reports with crop and pastureland maps, we calculated the number of control reports within each 1 km × 1 km grid cell for cropland/rangeland analysis and 10 km × 10 km grid cell for crop production analysis. All analysis and map preparations were performed using R Statistical Software (version 4.1.0; R Foundation for Statistical Computing, Vienna, Austria).

# 3. Results

## 3.1. *Temporal Distribution of Locust Reports and Control Operations*

The compiled dataset comprised more than 75,000 desert locust records with 42.5% reported in East Africa, 23.3% reported in the Middle East, and 34.2% reported in Eastern Region (Figure 1). Desert locusts can be either in solitarious (hoppers and adults) or gregarious (bands of hoppers and swarms) phases. In addition, remnant locusts from control operations against hopper bands and swarms were sometimes reported as gregarious hopper and adult individuals or groups. This is not unusual during upsurges. In terms of the types of desert locust classified within the reports, 19.7% were swarms, 14.3% were bands, 39.4% were solitarious adults and 26.6% were solitarious hoppers. There was a high degree of temporal heterogeneity in the numbers of reports. The highest weekly number of swarm reports (855) was reported in East Africa on the week commencing the 26 January 2021, the highest number of band reports (802) was also reported in East Africa on the week commencing the 9 June 2020. This coincides with the breeding periods.
<figure><img src="image_100.png" /><figcaption>**Figure 1.** The weekly numbers of desert locust reports. Surveys are categorised as swarms, bands, adults or hoppers. Data from [24] (LocustHub).</figcaption></figure>

The number of control operations performed in 2019–2021 reflects the enormous scale of the desert locust upsurge and deployment of resources to manage it (Figure 2). Overall, more than 39,000 control operations were conducted with 28.7% reported in East Africa, 23.8% reported in the Middle East and 47.5% reported in the Eastern Region. These consisted of 66.9% vehicle based control, 8.5% air based control and 4.8% handheld based control. About 19.8% of control reports were classified as “Unknown”. This is likely to be control by teams using digital tools that report only basic information, e.g., control = Yes/No without indicating control method.

<figure><img src="image_101.png" /><figcaption>**Figure 2.** The weekly number of control operations. Control operations are categorised according to region: East Africa, the Middle East and the Eastern Region. Data from [24] (Locust Hub).</figcaption></figure>
An increasing trend in the application of aerial control operations can be seen, with the peak reaching around 100 weekly aerial operations in November 2020 and February 2021. Most of the aerial control operations (86.2%) were conducted in East Africa. Further, within East Africa, 64.8% of aerial operations were conducted in Ethiopia, 4.7% in Kenya and 19.9% in Somalia. Overall, Kenya had the largest number of control reports (92.4%) classified as "Unknown".

The intensity of control operations differed amongst the regions (Figure 2). In all regions, the peaks correspond to breeding periods and are a result of an increase in locust numbers. In East Africa, the peak of control operations was in July 2020 (around 500 weekly control operations), followed by an approximately constant number of control operations (around 100 per week) from October 2020 till April 2021. In the Eastern Region, there were two peak periods reaching around 600 weekly operations in September–November 2019 and 700 weekly operations in May–August 2020. The desert locust situation was brought under control in the Eastern Region in Iran, Pakistan, and India in September 2020 [30]. In the Middle East, the intensity of control operations varied, a few periods (June 2019, January 2020, and May 2020) reached 300 operations.

In terms of pesticides applied during control operations, Malathion was frequently used in East Africa (18.5% of the reports) and the Eastern Region (65.8% of the reports). Malathion is an organophosphorus compound with a high kill rate for desert locust [31]. Malathion has low persistence in the environment as it breaks down quickly due to hydrolysis, photolysis and biodegradation [32]. The most frequently used pesticides in the Middle East were Deltamethrin (38.3% of the reports) and Fenitrothion (35.8% of the reports). Deltamethrin is a synthetic pyrethroid having low toxicity to birds and mammals [33]. Fenitrothion is an organophosphate, which has been shown to have a strong and long-lasting effect on non-target insect taxa [34]. Metarhizium, which is a biopesticide and can induce high mortality in S. gregaria [35], has been extensively applied in Somalia and used to treat at least 76.5% of all controlled area. Pesticide usage was ranked as follows according to area treated. In East Africa: Malathion (28.5%), Chlorpyrifos (17.5%), Metarhizium (6.0%), Fenitrothion (3.2%), Teflubenzuron (1.2%), Deltamethrin (0.3%), Triflumuron (<0.01%), Lambda-cyhalothrin (<0.01%). In the Eastern Region: Malathion (40.7%), Deltamethrin (38.8%), Lambda-cyhalothrin (11.3%), Fenitrothion (0.8%), Diflubenzuron (0.05%), Metarhizium (0.02%), Chlorpyrifos (<0.01%). In the Middle East: Fenitrothion (48.1%), Deltamethrin (33.8%), Malathion (10.4%), Chlorpyrifos (2.7%), Lambda-cyhalothrin (1.9%), Diflubenzuron (0.5%), Triflumuron (0.05%), Teflubenzuron (0.05%), Metarhizium (0.03%).

We found high correlations between the weekly number of desert locust reports and the weekly number of control operations: 0.94 in East Africa, 0.88 in the Middle East and 0.98 in the Eastern Region (Figure 3). However, there were differences between the three regions when we calculated the correlation coefficient between the number of swarm reports and control operations: 0.62 in East Africa, -0.02 in the Middle East and 0.74 in the Eastern Region. In East Africa, the highest correlation between the weekly number of different types of desert locust was between hopper reports and band reports (0.88). Low correlations between swarm reports and band reports were observed in all three regions: 0.01 in the Middle East region, 0.18 in East Africa and 0.27 in the Eastern Region.
<figure><img src="image_102.png" /><figcaption>**Figure 3.** Correlation matrices between the weekly number of control operations and desert locust reports. Colour intensity and the size of the circle are proportional to the correlation coefficients. Data from [24] (Locust Hub).</figcaption></figure>

## 3.2. Land Cover Characteristics

Figure 4 shows the frequency of land cover types for all regions and types of control. Bare parse vegetation, shrubs, cropland and herbaceous vegetation accounted for 96% of pixels with control operations. Bare/sparse vegetation is classified as lands with exposed soil, sand, or rocks and lands which never has more than 10% vegetated cover during any time of the year. Shrubs are classified as woody perennial plants with persistent and woody stems, or without any defined main stem, being less than 5 m tall [25].

The majority of vehicle based operations in the Middle East were reported over bare/sparse vegetation (83%), this is to be expected given that most of the region is desert. In the Eastern Region, a similar proportion of vehicle based control operations were reported over bare/sparse vegetation, shrubs and cultivated vegetation. In East Africa, 46% of aerial control were reported over land classified as shrubs, 20% over bare/sparse vegetation, and 14% over croplands. Overall, croplands (i.e., cultivated and managed vegetation/agriculture) accounted for 19% of the locations of control operations.

We could match 39.4% of control operations to swarm or band reports. The proportion of control operations that were not matched to swarms or bands (classified as 'NA' in Figure 4), could be explained by matching the control operations to solitarious locust reports (i.e., adult and hopper reports within a 1 km radius and up to two days before the control operation). This could be caused by erroneous classification of reports (for example, reporting remnant gregarious locusts from control operations as adults), as the standard operational procedure is that no sites with solitarious locust presence are subjected to control operations.

In East Africa, swarm reports preceded 35% of aerial operations conducted over bare/sparse vegetation, 65% of aerial operations conducted over shrubland, and 58% of aerial operations conducted over cropland.

## 3.3. Damage to Crop and Pasture Areas

We mapped the locations of control operations and desert locust reports against two of the factors determining livelihood: the proportion of grid cell area used as cropland and the proportion of grid cell area used as rangeland. We found that 13.3% of the grid cells with desert locust presence and 13.2% with control operations had a proportion of croplands higher than 0.75 (Figure 5). Similarly, 16.1% of the grid cells with desert locust presence and 14.9% with control operations had a proportion of rangeland higher than 0.75 (Figure 5).

To identify which crops are at the highest risk due to the desert locust upsurge, we used the data from the 2010 Spatial Production Allocation Model (mapSPAM) [28] to estimate crop production for the pixels with control operations. In East Africa, sugarcane (854 kt), other root crops (601 kt), maize (588 kt), sorghum (473 kt) and wheat (306 kt) were the five most vulnerable crops in terms of frequency of reported swarms and bands. In
the Middle East, the top two crops potentially affected by desert locust were vegetables (1720 kt) and wheat (666 kt). In the Eastern Region, Wheat (4360 kt) and sugarcane (3923 kt) were the two most infested crops. Overall, wheat production would be under the highest strain (5151 kt) due to desert locust infestations.

<figure><img src="image_103.png" /></figure>

**Figure 4.** Relationship between control type, locust reports and land cover in the three regions. Y axis corresponds to the number of reported control operations. The most frequent land cover types are given by bare/sparse vegetation (Bare veg.), cropland, herbaceous vegetation (Herb. veg.) and shrubland. Locust reports refer to the gregarious categories ‘Swarm’ and ‘Band’ that were matched to reports of control (i.e., reports within a 1 km radius and up to two days before the control operation in the Locust Hub dataset [24]. The ‘NA’ (‘Not Assessed’) category refers to control operations that could not be matched to the gregarious classes.

<figure><img src="image_104.png" /></figure>

**Figure 5.** The fraction of land used as cropland and rangeland for pixels with locust reports and control operations. It should be noted that timing of locust infestations with crop production and pasture availability are not reflected in the above maps.

We used mapSPAM data for Sub-Saharan Africa (v2 2017) [29] to investigate crops that were at risk from desert locust presence in East Africa at a national level. In Ethiopia, the top five crops affected were other roots (451 kt), sorghum (437 kt), maize (418 kt), wheat (354 kt) and other cereals (339 kt). In Kenya, the largest effect was on maize (267 kt), vegetables (186 kt) and potato crops (143 kt). In Somalia, the most affected crops were sorghum (8 kt),
maize (8 kt) and groundnut (5 kt). Overall, in the three countries, maize (694 kt), sorghum (454 kt) and other roots (453 kt) were the top three crops at the highest risk.

**4. Discussion**

Desert locust surveys and control operations are two management actions that have direct influence on desert locust dynamics [15]. More than 73,000 desert locust records and more than 38,000 control operations have been reported during the 2019–2021 desert locust upsurge. We found high correlation (0.92–0.98) between the weekly number of desert locust reports and the weekly number of control operations.

Control operations in the three regions were qualitatively different in terms of the type of control used for locust management. In East Africa 25.8% of control operations were aerial and 21.5% were vehicle based, while a variety of pesticides were used as control with the most frequent being Malathion (18.5%). In the Middle East 89.5% of control operations were executed by vehicle, and most frequently used pesticides were Deltamethrin (38.3% of the reports) and Fenitrothion (35.8% of the reports). In the Eastern Region 84.5% of control operations were vehicle based, and Malathion was most frequently used pesticide (65.8% of the reports).

Desert locust behaviour and dispersal patterns are complex processes that depend on environmental factors such as temperature [36], precipitation [22,37], the condition of vegetation [38–40], soil moisture [41,42], soil composition [41] and the wind [36]. These factors can create either stimulatory or inhibitory conditions for gregarisation, desert locust migration and breeding. For example, low-level wind-field convergence and concentration within the preferred habitat of desert locusts can affect the rate of gregarisation [43,44], whilst sandy soil is required for female desert locusts to breed [41]. Our analysis shows that the majority of control operations (79% in the Middle East, 28% in East Africa and 24% in the Eastern Region) were conducted over bare/sparse vegetation; this land type has high potential for the sufficient sand and clay content required for breeding. It is also the typical habitat of desert locust, which differentiates them from typical pests found in crops.

Despite improvements in both preventative control techniques and international collaboration in desert locust prevention, the desert locust remains a major threat to sustainable agriculture and rural livelihoods [7]. Desert locust infestations can aggravate food shortage situations [45]. We found that 13.3% of the grid cells with desert locust presence and 13.2% of the grid cells with control operations had a proportion of croplands higher than 0.75. We found that maize, sorghum and other roots were the top three crops at the highest risk in Ethiopia, Kenya and Somalia. Overall, our analysis of the Locust hub data indicate that wheat production is under the highest strain due to desert locust invasions in the 2019–2021 upsurge. This is in agreement with the estimate of a 12% decline in productivity of wheat due to the current desert locust upsurge in India [46].

Rangeland damage by desert locusts is also a threat to rural livelihoods. Rangelands provide local rural communities with natural resources for livestock production as grazing areas, as well as contributing to their livelihoods with fuel wood, poles, medicinal plants and wild foods. For example, for the most recent data available in 2019, Somalia was the largest producer of camels in East Africa, with the total number of camels recorded 7,243,792 [47], and a mean increase in camel population of 1.77%/year [48]. Rangelands also deliver important ecosystem services such as maintaining biodiversity and storing large amounts of carbon [49]. We found that 16.1% of the grid cells with desert locust presence and 14.9% of the grid cells with control operations had a proportion of rangeland higher than 0.75. Therefore, our analysis confirms that control operations continue to be used both in the reduction of desert locust populations and protection of crops to limit damage. Further work is required, however, to assess the cost effectiveness of surveillance and deployment of chemical control in reducing the increase and spread of locust populations during an upsurge, and also in managing agricultural and rangeland losses. It is possible that optimal deployment strategies of where to survey and where to apply chemical control may differ for minimising crop and other losses, compared with minimising insect
dispersal. Moreover, the relative cost-effectiveness and availability of aerial, vehicle-based and hand-held control needs to be assessed as does the environmental and human health impacts of different pesticide options relative to the obvious needs for food security.

Pest outbreaks can strongly affect local economies with invasive insects costing an estimated minimum of US$70billion per year globally [50]. Epidemiological modelling has been regularly used as a tool to evaluate different strategies for crop protection from pests and diseases [51]. Typically, model development requires the partition of spatial data into layers such as 'host/habitat layer', 'environment layer', 'pest layer', and 'control layer' [52]. A spatially explicit model of pest entry and dispersal can be combined with these heterogeneous layers to identify the outcome of alternative control strategies. Furthermore, control strategies can incorporate time-varying economic constraints [53] or human and social dimensions [54]. We anticipate that the present study will help to develop a novel epidemiological modelling framework incorporating optimal control theory for deployment of scarce resources [55,56] to inform the surveillance and management of desert locust infestations.

**Author Contributions:** Conceptualization, R.R., R.G.K.H. and C.A.G.; methodology, R.R. and C.A.G.; software, R.R.; formal analysis, R.R.; investigation, R.R., R.G.K.H., K.C. and C.A.G.; resources, C.A.G.; data curation, K.C. and R.R.; writing—original draft preparation, R.R., R.G.K.H., K.C. and C.A.G.; writing—review and editing, R.R., R.G.K.H., K.C. and C.A.G.; visualization, R.R.; funding acquisition, C.A.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by The UK Foreign, Commonwealth and Development Office. CAG also acknowledges support from the Bill and Melinda Gates Foundation.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data are publicly available from the sources listed in Materials and Methods.

**Acknowledgments:** The authors gratefully acknowledge Alexandre V. Latchininsky (FAO, Plant Production and Protection Division (NSP), Rome) for critical reading of the manuscript and his comments.

**Conflicts of Interest:** The authors declare no conflict of interest. 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.

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Article

# Long-Term Decline in Harvester Termites in Madagascar following Multiple Barrier Treatments with Fipronil against Migratory Locust

Ralf Peveling

Department of Environmental Sciences—Geoecology, University of Basel, 4056 Basel, Switzerland; ralf.peveling@unibas.ch

**Abstract:** Mounds of the harvester termite *Coarctotermes clepsydra* (Sjöstedt) (Isoptera: Termitidae, Nasutitermitinae) are a typical feature of savanna woodlands in Madagascar. With densities of up to 300 termitaria ha⁻¹, this species provides key ecosystem services and is an important food source for wildlife. Following large-scale aerial blanket and barrier treatments with the insecticide fipronil to control an outbreak of migratory locust in the late 1990s, evidence emerged that *C. clepsydra* and related food webs were adversely affected. However, neither the scale nor the duration of the effects were known. The present ex post study investigated the recovery of *C. clepsydra* populations subjected to multiple barrier treatments against hopper bands in 1998 and 1999 at estimated cumulative dose rates of 1.7–3.4 g fipronil ha⁻¹. At the time of the survey in 2007, both the density of occupied termitaria (30.2 versus 106.8 mounds ha⁻¹) and mound occupancy (24.3% versus 70.0%) were significantly lower in repeatedly sprayed so-called hotspots than in unsprayed areas. The overall adverse effect (mortality in sprayed areas corrected for mortality in unsprayed areas) was *E* = 64.4%. The main outcome of this study is a strikingly low resilience of *C. clepsydra* populations, which did not recover in hotspots within eight years, with likely repercussions on food webs. This study shows that the environmental benefits of barrier treatments are forfeited if the same areas are treated repeatedly during the same campaign. Recommendations are given for the mitigation of these risks.

**Keywords:** insecticide disturbance; barrier treatment; spray history; non-target effects; *Coarctotermes clepsydra*; recovery; resilience

## 1. Introduction

Madagascar is a global biodiversity hotspot and has a high level of endemism in most taxonomic groups. Termite diversity, in contrast, is low [1], with only 56 species known in 2003, with the majority of them being wood feeders [2]. The grasslands and savanna woodlands in the semi-arid southwestern part of the island are home to just one mound-building species: the grass-feeding harvester termite *Coarctotermes clepsydra* (Sjöstedt) (Isoptera: Termitidae, Nasutitermitinae), whose conspicuous conical-shaped termitaria can reach densities of 100–300 mounds ha⁻¹ [2,3]. *Coarctotermes* is one of three grass-feeding genera in the Nasutitermitinae and the only one in Madagascar [4]. Given their high biomass and exposed lifestyle during nocturnal grass-harvesting bouts, harvester termites are an important food source for both invertebrates [5] and vertebrates [6]. In Madagascar, they are also commonly collected for chicken feed [7,8].

Harvester termites share their habitat with the Malagasy migratory locust, *Locusta migratoria capito* Saussure, and the red locust, *Nomadacris septemfasicata* (Serville) (Orthoptera: Acrididae), and are therefore at risk of exposure to locust insecticides during control operations. During the last two major locust invasions, which lasted from 1997–2000 and 2013–2016, aerial control operations extended over 42,000 and 23,000 km², respectively [9,10], relying mainly on synthetic chemical insecticides and, during the 1997–2000 campaign, on barrier treatments with fipronil.

**Citation:** Peveling, R. Long-Term Decline in Harvester Termites in Madagascar following Multiple Barrier Treatments with Fipronil against Migratory Locust. *Agronomy* **2022**, *12*, 310. https://doi.org/10.3390/agronomy12020310

Academic Editor: Marco Scortichini

Received: 10 October 2021

Accepted: 20 January 2022

Published: 25 January 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

<figure><img src="image_107.png" /></figure>

**Copyright:** © 2022 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Barrier treatments target marching hopper bands, which cannot fly. The technique is based on barriers of sprayed vegetation lying hundreds of meters apart. It is considered environmentally benign because large tracts of land remain untreated [11,12]. While traversing the area, feeding hopper bands eventually pass through treated vegetation and die. During the 1997–2000 campaign, fipronil, a relatively persistent phenylpyrazole locust insecticide and also a potent termiticide [13,14], was the sole control agent for barrier treatments used at an operational scale [15]. Overall dose rates (i.e., rates applied over the total target area) of single barrier treatments ranged from 0.75–1.50 g active ingredient (a.i.) ha⁻¹ at a 1000–500 m barrier spacing, respectively. In heavily locust-infested areas, treatments were applied repeatedly within the same season, which accordingly led to higher cumulative dose rates.

Earlier studies have shown detrimental effects on non-target fauna of both single bar-
rier (1.0 g a.i. ha⁻¹) and blanket (3.2–4.0 g a.i. ha⁻¹) treatments with fipronil [16]. Harvester
termites were found to be particularly vulnerable, with their decline leading to a decline in
termite and/or ant-feeding reptiles and small mammals [3]. Field studies in Senegal and
Australia corroborated the adverse effects of blanket treatments on termites over a range of
dose rates (Senegal: 2.0–5.0 and 10.0–12.0 g a.i. ha⁻¹; Australia: 1.25 g a.i. ha⁻¹), though
these studies did not investigate food chain effects [17–19]. In contrast, a subsequent study
in Australia focusing on wood-eating termites, which owing to their more cryptic lifestyle
are less exposed to insecticides, found no hazards associated with barrier treatments using
fipronil at dosages per unit area ranging from 0.25–1.25 g a.i. ha⁻¹ [20].

While the fipronil-induced decline in harvester termites in Madagascar was unequivocal, eventually leading to cancellation of the registration in 2006 (Arrêté n°4196/06, 23 Mars 2006), its duration and spatial scale were unknown. Rapid assessments in southwestern Madagascar in 2005 provided circumstantial evidence of low densities of live colonies of *C. clepsydra* in former barrier treatment areas while abandoned and to varying degrees degraded mounds were abundant (author's observation). The present ex post study followed from this evidence. It tested the hypothesis that densities and occupancies of *C. clepsydra* termitaria and morphological features differ among unsprayed (no barrier treatments recorded) and sprayed areas subjected to multiple (≥2) barrier treatments from November 1998 to May 1999. The overall goal was to draw from the Madagascar experience and to inform risk assessments of fipronil and control agents with similar ecotoxicity worldwide.

**2. Materials and Methods**

This study was conducted in southwestern Madagascar. Based on spray protocols
from aircraft equipped with differential global positioning systems, all barrier treatment
areas (targets) were mapped on a monthly basis from November 1998 to May 1999 by the
European Union (EU) Food Security Programme in Madagascar [15]. The main reason
was to inform the campaign organization and to demonstrate progress in the control of
the invasion. Original shape files of the Geographical Information System were lost after
the campaign, but hard copies of the monthly spray maps and copies of the original spray
protocols for 33 (out of more than 200) barrier treatments were retrieved by the author at
the Delegation of the EU in Madagascar in 2005. The present study focused on an area
covering 32,180 km² where intensive control operations had taken place (Figure 1). The
area includes parts of the Sakaraha province (West) and the Haut Plateau de Horombé
(East) and the national parks of Zombitse Vohibasia (West) and Isalo (East). Spray areas
were, for the most part, adjacent to each other and sprayed only once, but a considerable
proportion overlapped, resulting in multiple treatments (≥2) within the same campaign.
The present study focused on these hotspots. The underlying assumption was that if full
recovery of harvester termites was achieved in hotspot areas, the same would hold for the
entire barrier treatment area.
<figure><img src="image_108.png" /><figcaption>**Figure 1.** Study area and barrier treatment areas (targets) in southwestern Madagascar. Areas sprayed at least three times (dark grey) are included in areas sprayed at least twice (grey) and the latter are included in areas sprayed at least once (light grey). The design included 9 randomly selected pairs of sites—or blocks—located in untreated areas (black circles) and repeatedly treated hotspots (white circles), respectively. At each site, the densities, occupancies, and morphologies of termitaria were measured. At the single site located in Isalo national park (black triangle), only mound morphologies were measured.</figcaption></figure>

According to the registered use of fipronil (Adonis® 7.5 UL) for barrier treatments in 1998, the maximum distance between barriers was 1000 m (Ministère d'Agriculture, Décision 166/98). No minimum distance was given at that time, but a decree issued by the Centre National Antiacridien in 2005 stipulated that the inter-barrier space should be 10 times as wide as the barrier (Note de service n°117-05/MAEP/CNA/Dir). According to available spray protocols (N = 33), the median barrier spacing was 875 m (range: 500–1000 m). At this distance, cumulative overall dose rates of fipronil correspond to ≈0.9, 1.7, 2.6, and 3.4 g a.i. ha⁻¹, respectively, at 1 single or 2, 3, and 4 overlapping barrier treatments. These values, however, are proxies because the actual combination of barrier spacing and the number of treatments was unknown for most targets.

A randomized paired-sample design was adopted, in which each site located in the center of a multiple barrier-treated hotspot was paired with a site located in an unsprayed area at a distance of 15–20 km (Figure 1). Sites were selected as follows: maps of barrier spray areas were scanned and imported into ArcGIS 9.4 (Esri Company, Redlands, CA, USA). They were georeferenced using the coordinates of targets for which spray protocols were available. Maps depicting monthly spraying operations were overlaid to identify overlapping areas of targets (Figure 1). Sampling sites were arbitrarily placed in the approximate center of hotspots. Paired unsprayed sites were arbitrarily placed in adjacent unsprayed areas at a minimum distance of 2–3 km from the boundary of the nearest sprayed area. For the purpose of the study presented in this paper, 9 pairs of sites—or blocks—were selected. The farthest distance between blocks was more than 200 km (Figure 1). Before starting the field work, the selected sites were verified to be located in typical locust and harvester termite savanna habitats by overlaying the barrier treatment map on the digital land use map of Madagascar (BD 500, Zone UTM 38 k, Institut Géographique et Hydrographique National de Madagascar, Antananarivo, Madagascar). The fire-resistant spear grass (*Heteropogon contortus*) is by far the most dominant grass species in these fire-prone savannas [21].
Each site was composed of 5 plots measuring 1000 m² each. The plots marked the center (original coordinates) and 4 corners of 1.4 × 1.4 km quadrats, with the corners located at a distance of 1 km at angles of 30°, 120°, 210°, and 300°, respectively, from the center point. Of the designated five plots per site, four were intended to be sampled depending on accessibility. Another site comprising six plots was investigated in Isalo national park. This was not planned before. Termitaria located there offered the opportunity to measure mound morphologies in presumably undisturbed conditions (no insecticide use, no harvesting of termites by humans).

The field work was conducted in March 2007. The designated plots were approached by vehicle as close as possible and further on foot, using the go-to function of the Global Positioning System. The median deviation from target waypoints was 7 m. A 17.84 m rope with wooden pegs fixed to both ends was used to delineate plots. One peg was driven into the ground to mark the center. The rope was pulled to its full length and the opposite peg circulated to outline the perimeter of a circle measuring 35.68 m in diameter and 1000 m² in surface area. All termitaria or remnants thereof lying within the circle were investigated. Even fully degraded or overgrown termitaria could be identified because grasses grew taller on the nutrient-enriched soil and subterranean mound chambers were still detectable. Mounds on the boundary were included if the center lay within the circle. The following parameters were recorded: minimum and maximum basal diameter, maximum height and number of domes, mound occupancy, and traces of human use such as regularly shaped marks from spades used to dig up termites. Neighboring termitaria with basal diameters >1 m apart were treated as different colonies. To verify occupancy, mounds were probed using picks. The search was extended to subterranean parts if no specimens emerged. Specimens of workers and soldiers were stored in 80% alcohol.

Data from the four plots per site (only three plots in one site) were pooled and extrapo-
lated to one hectare. Mound density data were log-transformed and percent occupancy data
(equivalent to survival rates) arcsine-transformed prior to two-factor analysis of variance
(ANOVA) without replication, with treatment as the fixed and block as the random factor.
This analysis is similar to a paired sample t-test.

The adverse effect *E* (mortality in repeatedly sprayed areas corrected for mortality in unsprayed areas) was calculated as a descriptive statistic according to Schneider-Orelli [22], where the percentage mortality per site was calculated as 100% minus the survival rate in this site. Effect calculations normally require pre-spray data from both sprayed and unsprayed sites. These were obviously not available as this study was conducted at 1 point in time 8 years post-spray. However, because mound structures persist for many years after the death of a colony, pre-spray densities (i.e., the status ante) of harvester termite colonies could be approximated.

The mound characters of occupied termitaria were analyzed using one-way ANOVA.
Basal diameter was calculated as the mean of the minimum and maximum values. Data
were log+1-transformed to achieve homogeneity of variances as needed. Analyses included
pooled data from repeatedly sprayed, unsprayed, and control (national park) areas. Differ-
ences among means were tested using the Student–Newman–Keuls (SNK) test. A two-way
contingency table was used to examine the association between treatment (repeatedly
sprayed versus unsprayed) and termite-collecting efforts (traces of human use versus no
traces). All tests were conducted using PASW Statistics 18 (SPSS Inc. Hong Kong, China).

3. Results

3.1. *Density and Occupancy of Termitaria*

The mean density of combined occupied and abandoned termitaria in repeatedly
sprayed areas (93.5 ± 17.1 mounds ha⁻¹, mean ± SE) was significantly less than that
recorded in unsprayed areas (156.6 ± 26.4 mounds ha⁻¹; F₁,₈ = 9.1, *p* = 0.017). Likewise,
the mean density of occupied termitaria was significantly less than in unsprayed areas
(30.2 ± 15.1 ha⁻¹ versus 106.8 ± 18.6 mounds ha⁻¹; F₁,₈ = 17.9, *p* = 0.003) (Figure 2a). With
≤3 inhabited termitaria ha⁻¹, 4 out of 9 barrier treatment sites were virtually void of live
harvester termite colonies though remnants of termitaria were frequently found. Mound
occupancy was 24.3 ± 8.8% (mean ± SE) in repeatedly sprayed areas and 70.0 ± 4.7%
in unsprayed areas (F<sub>1,8</sub> = 15.2, *p* = 0.005) (Figure 2b). The resulting adverse effect was
*E* = 64.4 ± 12.7% (mean ± SE), meaning that at nearly 8 years post-spray, populations were
suppressed by this percentage.

<figure><img src="image_109.png" /></figure>

**Figure 2.** Boxplots of the density (a) and percentage (b) of occupied termitaria in unsprayed (no treatment recorded) and repeatedly sprayed (multiple barrier treatments) areas (N = 9 in both treatments). Box plots show the median, lower/upper quartile, and smallest/largest observation and outliers (○). Differences among sprayed and unsprayed areas are statistically significant (see text).

3.2. Mound Morphology

The height of the occupied mounds was similar in sprayed (24.3 ± 1.8 cm; mean ± SE) and unsprayed (22.1 ± 0.9 cm) areas but differed significantly from the height recorded in undisturbed conditions in the Isalo national park (49.3 ± 4.5 cm; *F*<sub>2,524</sub> = 14.9, *p* < 0.001, SNK test significant at *p* < 0.05) (Figure 3a). Mound basal diameters were also similar among repeatedly sprayed (68.6 ± 3.9 cm) and unsprayed (70.7 ± 2.0 cm) areas but smaller than in undisturbed conditions (117.6 ± 8.1 cm; *F*<sub>2,524</sub> = 20.5, *p* < 0.001, SNK test significant at *p* < 0.05) (Figure 3b). The maximum number of domes per colony was 2. The mean number was 1.06 ± 0.04 in the national park and 1.07 in both unsprayed (SE = 0.01) and repeatedly sprayed (SE = 0.02) areas. These differences were not significant (*F*<sub>2,455</sub> = 0.02, *p* = 0.980).

3.3. Human Use of Harvester Termites

Of all the occupied mounds in unsprayed areas (N = 377), 33.4% showed traces of anthropogenic activity. In most cases, the apex (where termites concentrate in the morning) had been cut off, resulting in a flat- or bowl-shaped top after repair. In repeatedly sprayed areas (N = 119), the percentage was only 17.6%, which was significantly less than expected (29.6%; N = 496, d.f. = 1, χ² = 10.8, p = 0.001).
<figure><img src="image_110.png" /><figcaption>**Figure 3.** Boxplots of the height (a) and mean diameter (b) of occupied termitaria in unsprayed (N = 377; no treatment recorded) and repeatedly sprayed (N = 119; multiple barrier treatments) areas and in a national park (N = 31). Box plots show the median, lower/upper quartile, smallest/largest observation, outliers (○) and extreme cases (*). Means with different small letters are significantly different.</figcaption></figure>

**4. Discussion**

The main outcome of the present research is a strikingly low resilience of *C. clepsydra* populations in hotspot areas. When exposed to 2-4 barrier treatments of fipronil within a single locust campaign, populations did not recover to pre-spray levels after 8 years. Large areas of intensive control were virtually cleared of *C. clepsydra*. This low resilience is likely due to the low dispersal capacity of termites. Dispersal distances generally do not exceed a few hundred meters [23] because alates are poor flyers [24]. Recolonizing vast deserted areas through repeated cycles of colony founding, growth, and dispersal would take a long time, depending on the density and spatial distribution of source colonies, and possibly also on the availability of abandoned but intact mounds for easier recolonization.

Another important outcome is that the environmental benefits of barrier over blanket treatments [11,12] are jeopardized if the same areas are sprayed repeatedly. The same applies if the barrier spacing is too narrow to provide truly unsprayed refugia from where recolonization can start. In an earlier study, even a 750 m barrier spacing resulted in a nearly 50% suppression in harvester termites after 2 years [3]. Considering that >30% of 33 barrier treatments for which spray protocols were available used a barrier spacing of only 500 m, adverse effects likely extended well beyond the 436 km² multiple barrier treatment hotspots studied in the present study. Extrapolating to the study area (Figure 1), 30% would correspond to about 3500 km². However, further research on *C. clepsydra* populations in single barrier treatment areas would have been required to test and quantify this assumption.

In any case, long-lasting reductions of harvester termites are expected to result in cascading effects across the trophic levels of affected ecosystems. This is because termites provide an array of ecological services, including decomposition of organic matter, nutrient cycling, enhancement of soil drainage, or bioturbation [25-28]. Moreover, termites influence the composition, structure, and spatial variation of vegetation [27,29,30] and the dynamics of ant communities [31], and mound-building termites play a keystone role by creating habitats for inquiline invertebrates [32]. The extent to which any of these processes was upset by the decline in *C. clepsydra* is not known. However, given its unique position as the only mound-building and grass-harvesting species of the savanna woodlands of Madagascar, perturbations were likely to be ecologically significant. At higher trophic levels, indirect effects of fipronil on wildlife are mediated mainly through loss in the quantity or quality of prey [33]. The above-mentioned study in Madagascar showed that the decline in harvester termites resulted in a reduced abundance of termite-feeding lizards and tenrecs [3]. A long-lasting decline in harvester termites over large areas would, therefore, affect the entire food web to which they belong.
In sub-Saharan Africa, termites and termite mounds are widely utilized by humans [8,34]. Examples include the consumption of protein-rich alates [35] or the use of mound material as fertilizer [36] or plaster for traditional houses [37]. In Madagascar and many other African countries, termites are frequently collected for chicken feed [7,8]. *C. clepsydra* colonies appear to recover well from recurrent damage to their mounds and loss in biomass, though this study showed that termitaria in both sprayed and unsprayed areas were smaller than in a protected area where the harvesting of termites is not allowed. An interesting finding was that inhabited termitaria in repeatedly sprayed areas were less frequently harvested than those in unsprayed areas. This suggests that it is less profitable to exploit a more widely dispersed resource because it requires more time and longer distances to find live colonies. It also shows that adverse effects on harvester termites directly translate into a negative impact on rural livelihoods.

An important methodological feature of this study was that inferences were made on pre-spray termitaria densities by combining occupied and abandoned termitaria. This was possible because remnants (e.g., subterranean mound chambers) or indications (e.g., circular grass patches different from surrounding grassland) of deserted mounds are detectable for many years. Moreover, the preponderance of deserted mounds in sprayed areas indicated that these had indeed been harvester termite habitats in the past.

However, the observed lower density of combined occupied and abandoned mound densities in repeatedly sprayed areas suggests that some fully weathered termitaria may have been undetected at the time of the field work. In this case, pre-spray densities and hence the adverse effect *E* would have been underestimated. There are only few studies on the persistence of abandoned mounds. A 10-year study on the growth of the Australian harvester termite *Drepanotermes perniger* (Froggatt) (Termitidae, Termitinae) showed that deserted mounds were not any more eroded than inhabited ones [38]. Lobry de Bruyn [39] hypothesized that mounds of *Drepanotermes tamminensis* (Hill) take at least 30 years to erode to ground level. However, the outer wall of *Drepanotermes* mounds is thicker and harder than that of *Coarctotermes* mounds (author's observation). Furthermore, mound erosion in southwestern Madagascar would be expected to be faster than in the Australian outback owing to much higher rainfall (≈750 mm).

Conversely, the absence of epigeous mounds was considered an indication of the absence of harvester termites. This was reasonable because successful recolonization would have resulted in the development of visible mounds. Colonies of the ecologically similar harvester termite *Trinervitermes trinervoides* (Sjöstedt) (Nasutitermitinae) in South Africa begin building mounds 2–3 years after colony founding [40]. In view of this, significant visible recolonization would have been expected in sprayed areas after eight years.

Another question is whether wildfires may have had an effect on the outcome of this study. It is estimated that 450,000 ha of savanna are burnt in Madagascar each year [41]. However, there is no evidence that wildfires destroy colonies of harvester termites. On the contrary, grasslands dominated by fire-resistant spear grass are typical habitats of harvester termites. This is in line with studies in South Africa [42], Brazil [43], and Australia [44]. If wildfires supposedly have no effect on harvester termites, the reverse is not the case. With the densities of colonies and concomitant grass removal rates reduced, a larger proportion of the standing crop would be devoured by wildfires instead of being decomposed and recycled by termites.

**5. Conclusions**

Systemic insecticides, such as fipronil, have been linked to a variety of direct and indirect effects on vertebrate and invertebrate non-target organisms and ecosystems [33,45]. Insecticides that have adverse effects on populations of non-target invertebrates over prolonged periods are classified as high-risk products [11,46]. The results of the present and previous studies [3,16–18] leave no doubt that fipronil belongs to this category and that even barrier treatments can have long-lasting adverse effects on termites and related food webs. Therefore, rigorous and effective risk mitigation measures must be applied.
Apart from using less environmentally harmful insecticides for barrier treatment in the first place (e.g., benzoylurea insect growth regulators), risk mitigation measures should at least include the following: (i) mapping of historical and current locations of locust populations as a basis for improved monitoring, control, and impact assessment; (ii) monitoring of the magnitude and duration of adverse effects; (iii) restricting the use of fipronil or insecticides with similar ecotoxicity until full recovery of the affected non-target fauna has been evidenced; (iv) ensuring that the barrier spacing is wide enough to provide truly unsprayed refugia; and (v) imperatively avoiding multiple treatments.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the
corresponding author.

**Acknowledgments:** I am grateful to Christian Rejela and Christale Robelle Razafindrahova for sup-
porting the field work and to Clarah Andriamalala and Palvanijaz Pirnijasov for conducting GIS
analyses. The Centre National Antiacridien (CNA) of Madagascar is acknowledged for providing
logistical support and the EU Food Security Programme together with the Projet de Lutte Antiacridi-
enne dans l'Aire Grégarigène for mapping barrier treatments during the 1998/99 campaign. I am
also grateful to Harold van der Valk for his encouragement to undertake this study against all odds
and without external funding. The late Colin C.D. Tingle and his team members in Madagascar and
the UK are acknowledged for spearheading research on the environmental impact of fipronil and
other locust control agents in Madagascar.

**Conflicts of Interest:** The author declares no conflict of interest.

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<figure><img src="image_111.png" /></figure>

Article

# Linseed Oil Affects Aggregation Behaviour in the Desert Locust *Schistocerca gregaria*—A Potential Swarm Disruptive Agent

Zainab Ali Saad Abdelatti<sup>1,2 ID</sup> and Manfred Hartbauer<sup>1,*</sup>

Received: 17 August 2020; Accepted: 21 September 2020; Published: 24 September 2020

**Abstract:** Gregarious desert locusts constitute very destructive agricultural pests. They aggregate and form collectively moving swarms that devastate vegetation and reduce crop production. To combat gregarious locusts, a bio-pesticide formulation that contains linseed oil as the main component was described recently. Since linseed oil is rich in fatty acids, some of which function as necromones that indicate injury or death in various insect species, we investigated the influence of linseed oil on the aggregation behaviour of sexually mature gregarious desert locusts. For this reason, we performed a series of aggregation experiments with six individuals of the same sex and brushed the wings of one individual (target individual) with linseed oil. The time the oil brushed target males spent close to any other individual was reduced in 76% of trials (average reduction of 18%), whereas the time target females spent in groups with members of the same sex did not alter. These results suggest that linseed oil may act as a bioactive agent that has the potential to disrupt swarm formation.

**Keywords:** locust swarm; bio-pesticide; gregarious locusts; aggregation behaviour; linseed oil; necromones

## 1. Introduction

Desert locusts (*Schistocerca gregaria* Forsskål, 1775) are considered to be among the most serious agricultural pests because of their polyphagous feeding behaviour, rapid reproduction rates and quick migration patterns [1,2]. Like other locust species, this species displays density-dependent phase polyphenism, which means that they can transform reversibly between two phases in response to population density: The solitary and gregarious phases. Individuals in either phase differ in terms of their morphology, physiology and behaviour [1,3]. Behavioural changes can occur quickly, appearing within just a few hours [4]. If the population density is low, locusts exist in a solitary phase and avoid each other, except when they are ready to mate. If the population density increases, even over a short time period, the behavioural transition to the gregarious phase is evoked [5,6]. This is the case, for example, after rainfalls that end long periods of drought, resulting in nymphs hatching from eggs laid in burrows in the ground [7]. As the population density increases, the locusts become social, aggregate and form marching bands on the ground (wingless hoppers); later, they form collectively moving swarms that can migrate over long distances [1]. Locust swarms can devastate entire fields and cause extensive crop damage over very short time periods. A small swarm of locusts contains thousands of individuals that spread out over several hundred square metres, but large swarms contain up to 80 million individuals per square kilometre. Since such swarms can cover a distance of 100 km per day [1], farmers regard gregarious locusts as one of the most destructive plagues on earth.

<footnote n="1">¹ Institute of Biology, University of Graz, Universitätsplatz 2, 8010 Graz, Austria; zienabentomologist@gmail.com</footnote>

<footnote n="2">² Department of Zoology, Faculty of Science, South Valley University, Qena 83523, Egypt</footnote>

<footnote>* Correspondence: manfred.hartbauer@uni-graz.at; Tel.: +43-316-380-5615</footnote>
Locusts can sense swarm members using olfactory, visual and tactile cues [5,6,8]. Physical contact was found to be the most potent stimulus, causing solitary locusts to gregarise. Simpson et al. [8] discovered that touching the hind legs of others in a repetitive way induces phase transition. A patchy distribution of food plants also increases the probability of physical contact between locusts and boosts aggregation [9]. On the contrary, visual or olfactory stimulants have lesser or incompetent effects on phase transition in the desert locust [8,9], whereas the combination of them can lead to a behavioural gregarisation [10]. In addition, seeing other locusts for a longer period (24 h) can also mediate phase change, at least partially [10]. Agents that disrupt the formation of swarms have not yet been established on the market but may represent alternative measures that can be used against locust outbreaks.

The authors of this study recently developed a highly effective botanical pesticide formulation against two species of locusts that is mainly composed of linseed oil as a carrier oil and three essential oils [11]. A single spray treatment of locusts with this formulation killed all adults and nymphs of desert locusts (*Schistocerca gregaria*) and migratory locusts (*Locusta migratoria*) within 30 h [11,12]. In addition to this toxic effect, this botanical pesticide formulation may also exert a change in group formation behaviour after individuals have come in contact with linseed oil that contains 75–90% unsaturated fatty acids (50–55% linolenic acid, 15–20% oleic acid and 11–20% linoleic acid [13]). Yao et al. [14] described oleic and linoleic acid as necromones, substances that are associated with the injury and death of insects. These often evoke distinct behaviour patterns in other individuals of the same or different species. For example, eusocial and some semi-social species show necrophoric behaviour, including the removal of dead bodies from the nests (observed in bees, ants, spiders and aphids), burial (covering the dead with soil and/or other materials in ants and termites) and cannibalism (intraspecific necrophagy in ants and termites) [14–16]. In contrast, solitary and some sub-social species avoid dead or injured individuals (necrophobic behaviour) [14,16]. In this study, we performed aggregation experiments to test the following hypothesis: Linseed oil evokes a change in aggregation behaviour in gregarious desert locusts once one individual (“the target individual”) has come into contact with this oil. To test this hypothesis, we evaluated the time the target individual spent in close proximity to others in a group of six individuals before and after brushing its wings with linseed oil. Moreover, we tested the behavioural responses of desert locusts towards a stationary linseed oil target, dead and crushed locust bodies.

## 2. Materials and Methods

### 2.1. *Insect Species and Oil*

Desert locusts (*Schistocerca gregaria*) were purchased in the gregarious phase from a breeding stock provided by the Buchner Company in Austria. Locusts were maintained in a crowded colony at the Institute of Zoology in Graz. About 100 locusts were kept in a glass terrarium with the dimensions of 60 × 30 × 30 cm. The light:dark cycle was 12:12 h, and the average temperature in the terrarium was 28 °C at night and 35 °C during the day. The relative humidity was 45–60%. The individuals used in the behavioural experiments were of the same age (mature: about four or five weeks after their last moult).

Organic linseed oil (*Linum usitatissimum*, Natur-Pur brand) was purchased from Spar Österreich. It was stored in a dark bottle at 4 °C during testing.

### 2.2. *Insects Food*

The locusts' diet consisted of organic wheat seedlings and organic wheat bran (DM-Drogeriemarkt, Karlsruhe, Germany). Pots of wheat seedlings (i.e., “cat grass”) were purchased from Zoo Muser, a local pet shop. These pots of grass were watered daily and exposed to a light:dark schedule of 12:12 h. The wheat seedlings were replaced every two days in the terrarium.
## 2.3. *Experimental Setup*

Aggregation experiments were performed inside an anechoic chamber, using an arena with the dimensions of 85 × 65 × 45 cm. The floor of the arena was covered by a piece of paper that was replaced after each trial. We placed a heater (model: PF320LCD, ewt) to maintain a constant temperature of 30 °C inside the anechoic chamber. To illuminate the arena, we use two lamps, an 8 W inspection lamp (Electronic Montage Lamp, SLV Elektronik GmbH: Löhne, Germany) mounted on the top of the arena and a standard lamp (type: 160312, lamp/Bulb: 65× LED, 8 W, SLV Elektronik GmbH: Löhne, Germany). A top-view video camera (CB-38075, GKB: Taichung, Taiwan, China) was used to record the movement of locust individuals inside the arena.

## 2.4. *Insect Isolation*

Before performing aggregation experiments, we isolated gregarious desert locusts for three days to simulate swarm disruption that has to be expected after treatment of a swarm with the linseed oil based botanical pesticide. Therefore, we caged individuals in plastic boxes with dimensions of 9 × 9 × 6 cm and placed them at a distance of 2 cm from one another. Boxes were located near to a glass terrarium that contains crowded-reared locusts. All individuals used in experiments were taken from isolation boxes, behaved normally and were able to jump. The walls of insect boxes provided air exchange and olfactory communication among the individuals (ninety-eight pores with a diameter of 1 mm). These boxes were placed on sheets of soft kitchen paper towel to absorb the vibrations generated by the locusts. Additionally, white paper tape was applied to the walls of the plastic boxes to prevent visual contact. To study the aggregation ability of locusts three days after isolation, we evaluated the time individuals came close to any other individual in our aggregation experiments (see Table 1). During the isolation period, the same amount of grass was offered to all individuals. On the third day of isolation, a small piece of reflecting tape was mounted on the pronotum of one individual to mark the target locust. This reflecting tape was fixed in place using a small drop of super glue (Loctite, Henkel Central Eastern Europe GmbH, Vienna, Austria), which is harmless for insects such as bees and locusts.

## 2.5. *Behavioural Experiments*

Three days after their isolation, the locusts were transferred to the arena to study group formation during the light phase of their day cycle. In all trials, we studied the group formation of six individuals of the same sex to prevent premating and mating behaviour. All individuals were inspected for dual sexual characteristics (gynandromorph) to prevent an influence arising from mixing different sexes. The inter-individual distance between the target individual and any other individual was observed over a time period of 30 min before linseed oil application. Then, the wings of the target locust were brushed with a thin film of linseed oil (provided at room temperature), and the locusts were monitored for another 30 min. The movements of locusts were recorded under bright conditions using the top-view video camera. This camera was connected to a frame grabber (Pixelsmart Inc.: Lewiston, NY, USA) that captured images in intervals of 5 s, resulting in 12 images per minute. In total, 26 trials were performed with 13 female groups and 13 male groups. Since tactile stimulation may affect the target locust behaviour, we also performed sham operation experiments to exclude an influence of being handled and brushed. In sham operation trials, locusts were monitored over a time period of 30 min before treatment during the light phase of their day cycle. Then, the wings of the target locust were brushed with a clean brush, and the locust group was monitored for another 30 min after treatment. In total, 24 trials were performed with 6 female groups and 8 male groups. The ambient temperature and relative humidity were measured with a hand-held device during the trial. On average, the ambient temperature was 28.7 ± 0.9 °C (mean ± SD), and the mean relative humidity was 36.3 ± 2.9% (mean ± SD).
## 2.6. Behavioural Responses Towards Linseed Oil and Dead Insect Bodies

Experiments were performed on mature desert locusts of both sexes to study the attraction/avoidance behaviour after exposing locusts to linseed-oil-soaked paper and dead locust bodies. Five males or females were taken from the crowded-rearing arena and were placed in a test arena with a dimension of 48 × 24 × 28 cm. Two clean pieces of plastic foil (dimension of 8 × 3.5 cm) were placed on opposite corners of the arena. The movement of locust individuals was monitored for 30 min using a USB-camera that captured images in intervals of 5 s. Then, a filter paper saturated with 0.5 mL of linseed oil (dimension of 7 × 2.5 cm) was placed on a randomly selected plastic foil of the arena and locust individuals were monitored for another 30 min. Then, the filter paper was replaced by a clean plastic foil and the locusts were given 5 min pause before the next experiment was performed with dead insect bodies. For this purpose, two freshly killed locust individuals (a male and a female) were placed on the clean plastic foil opposite the linseed oil corner and the locust individuals were monitored for another 30 min. Locusts were killed by putting them into a freezer for one hour. In total, 11 trials were performed with 5 female groups and 6 male groups.

## 2.7. Behavioural Responses to Crushed Male Bodies

While intact dead bodies can be an indicator of contagion in wood lice, the crushed bodies of conspecifics can be a sign of an injury caused by a predator [14]. Therefore, we exposed male locusts to the crushed bodies of conspecific males in a similar setup. Two males were killed by freezing and then their bodies were crushed. A clean piece of plastic foil was placed in a randomly selected corner of the arena and the movements of locusts were monitored for 30 min by means of a top view USB-camera. Then, the foil was replaced by another foil to expose locusts to crushed conspecific males and to monitor locust movements for another 30 min. Ten trials were performed with groups of 5 males.

## 2.8. Data Evaluation and Statistical Analysis

The obtained video frames were imported into ImageJ (version 1.51j8, National institutes of Health: Bethesda, MD, USA) to measure the distances between the target locust and the next individual. Frame-by-frame distance measurements allow us to quantify the tendency to form groups. We regarded two or more individuals as a group when the inter-individual distance from the target individual was shorter than or equal to its body length (see red scale bar in Figure 1, on average: 5.8 cm for male groups and 6.2 cm for female groups). Such a small distance allowed individuals to see and smell each other. Gillet defined the grouping of locusts by the distance of two body-lengths to each other [17,18]. To analyse the group formation over a period of 30 min, 360 images were imported into ImageJ, and the time the target locust spent in groups (aggregation time) was evaluated by hand. The distance between the body of the target individual (see blue circle in Figure 1) and the closest individual was measured manually after spatially calibrating the image. The first five minutes at the beginning of frame grabbing (equals to 60 image) were excluded from the evaluation to remove possible behavioural influences related to insect handling. The time spent in groups was calculated using Equation (1):

$$Time \ spent \ in \ groups \ (minute) = \left( \frac{Sum \ of \ group \ formation \times 5}{60} \right) \qquad (1)$$

The term “sum of group formation” refers to the number of frames in which the inter-individual distance of the target individual from any group member was shorter than or equal to its body length. To calculate the number of minutes individuals spent in a state of aggregation, we multiplied the “sum of group formation” by 5, which refers to the inter-frame interval of 5 s, and divided the result by 60. Percentages of aggregation relative to an observation period of 25 min were calculated using Equation (2):

$$Probability \ of \ aggregation \ (%) = \left( \frac{Time \ spent \ in \ groups}{25} \right) \times 100 \qquad (2)$$
If the duration of group formation changed after the application of linseed oil or after sham operation by more than 10% (2.5 min), we considered this as a change in aggregation time. This threshold definition allowed us to discriminate between trials in which group formation either increased or decreased after linseed oil treatment.

To study the general tendency of the individuals to form groups after three days of isolation, the time any individual spent in groups of at least two individuals was quantified by measuring the distances between the closest parts of their bodies. We regarded two or more individuals as a group if their inter-individual distance was shorter than or equal to the average of the body lengths (on average: 5.6 cm for male groups and 6.2 cm for female groups) of all six locusts. The time any individual spent in groups was calculated according to Equation (1) (results are shown in Table 1).

To quantify the activity of the target individual before and after the application of linseed oil, the distance covered by the target individual was measured with the help of the MTrackJ plugin offered in ImageJ. The time spent in the target corner was measured by counting the frames in which any individual was within one body length to borders of the plastic foil. The time spent in the target corner was converted into minutes by using Equation (1).

The time spent by individuals in groups or in the target corner before and after the treatments was tested for statistically significant differences by performing a paired *t*-test. If data distribution deviated from a normal distribution, a Wilcoxon signed-rank test was performed. To test for statistically significant differences between the percentages of trials in which either an increased or decreased group formation time was observed after the application of linseed oil, we performed a z-test with Yates correction. All statistical tests were performed in Sigma Plot version 14 (Systat Software Inc.: Erkrath, Germany). The average amount of aggregation time and the proportion of aggregate formation relative to the observation period are given as the arithmetic mean ± standard deviation.

<figure><img src="image_112.png" /><figcaption>**Figure 1.** Snapshot of the top view camera. Blue circle: The target individual with the reflecting tape on the thorax. The red scale bar indicates the body length of the target individual, which was used as the minimum distance to define group formation.</figcaption></figure>

# 3. Results

## 3.1. *Group Formation after Isolation*

To study the general tendency of individuals to form groups after three days spent in isolation, we quantified the amount of time (see Equation (1)) during which **any individual** maintained a distance to other individuals that was equal to or smaller than the average body length of all locusts in a trial (Table 1). Neither males nor females changed the time spent close to any other individual before and after the wings of target individuals were brushed with linseed oil (females: 21.2 ± 2.2 min before
vs. 22 ± 2.7 min after treatment, *p* = 0.137, paired *t*-test, *N* = 13; males: 23.0 ± 2.1 min before vs. 21.8 ± 1.9 min after treatment; *p* = 0.112, paired *t*-test, *N* = 13).

<table><caption>**Table 1.** Time any individual spent in groups of at least two individuals (including the target one) before and after the application of linseed oil. Males and females were tested in separate experiments. Total observation time = 25 min, Group size = 6 individuals, *N* = 26.</caption><thead><tr><th rowspan="2">Experiments</th><th colspan="2">Time of Forming Groups of at Least 2 Individuals (minute)</th></tr><tr><th>Before Treatment</th><th>After Treatment</th></tr></thead><tbody><tr><td rowspan="10">Females</td><td>22.7</td><td>23.5</td></tr><tr><td>23.2</td><td>24.4</td></tr><tr><td>17.7</td><td>22.0</td></tr><tr><td>21.3</td><td>21.2</td></tr><tr><td>22.9</td><td>22.0</td></tr><tr><td>22.5</td><td>23.8</td></tr><tr><td>18.8</td><td>20.2</td></tr><tr><td>25.0</td><td>25.0</td></tr><tr><td>22.0</td><td>22.5</td></tr><tr><td>20.5</td><td>22.8</td></tr><tr><td>17.6</td><td>14.2</td></tr><tr><td>21.4</td><td>21.8</td></tr><tr><td>19.8</td><td>22.8</td></tr><tr><td>Average of females</td><td>21.2 ± 2.2</td><td>22.0 ± 2.7</td></tr><tr><td rowspan="10">Males</td><td>23.8</td><td>23.9</td></tr><tr><td>24.7</td><td>22.8</td></tr><tr><td>25.0</td><td>19.9</td></tr><tr><td>20.2</td><td>20.9</td></tr><tr><td>23.5</td><td>20.0</td></tr><tr><td>20.9</td><td>19.1</td></tr><tr><td>24.2</td><td>23.9</td></tr><tr><td>23.8</td><td>21.8</td></tr><tr><td>21.7</td><td>18.7</td></tr><tr><td>24.5</td><td>22.1</td></tr><tr><td>23.9</td><td>22.7</td></tr><tr><td>24.9</td><td>23.4</td></tr><tr><td>18.4</td><td>24.2</td></tr><tr><td>Average of males</td><td>23.0 ± 2.1</td><td>21.8 ± 1.9</td></tr><tr><td>Total average</td><td>22.1 ± 2.3</td><td>21.9 ± 2.3</td></tr></tbody></table>

## 3.2. Linseed Oil Treatment of Male and Female Groups

The average amount of time **target males** spent in groups with members of the same sex was significantly reduced by 4.6 ± 5.3 min (18.2 ± 21.2% of the observation time, Figure 2A, *p* = 0.010, paired *t*-test, *N* = 13) after brushing their wings with linseed oil. In 76.7% of trials, males reduced the time spent in aggregation by more than 10%. In these trials, the average aggregation time among males significantly decreased by 6.8 ± 3.2 min (27 ± 12.6% of observation time) after oil treatment (red horizontal stripes in Figure 2B, *p* < 0.001, paired *t*-test, *N* = 10). In only two trials did the
aggregation time among males increase by 4.9 ± 1.9 min (20 ± 7.8% of observation time, blue vertical stripes in Figure 2B), and no change was observed in one trial (black area in Figure 2B). Furthermore, the proportion of trials exhibiting a decreased aggregation time after treatment was significantly higher compared to the proportion of trials showing an increase (Figure 2B, *p* = 0.006, z-test, *N* = 13). Before the application of the linseed oil, the target males sometimes mounted other males or were mounted by other males. Interestingly, male–male mounting involving the target individual was never observed after the target locust’s wings were brushed with linseed oil. The average distance covered by the target males (activity) within 25 min did not change after linseed oil application (Table S1, *p* = 0.818, paired *t*-test, *N* = 13). Tracking brushed males revealed that, in 92.3% of trials, the target males were very active and moved towards other individuals in the arena, but other individuals were avoiding them.

<figure><img src="image_113.png" /><figcaption>**Figure 2.** Sex differences in group formation after linseed oil treatment. Time that the target males (A) and target females (C) spent in groups. Red lines: Time spent in groups decreased. Blue lines: Time spent in group increased. Black lines: Time spent in groups did not change by more than 10%. Total observation time = 25 min, *N* = 13 experiments. (B,D) Percentage of experiments in which aggregation time decreased (red horizontal stripes), increased (blue vertical stripes) or was left unchanged (black area) after linseed oil treatment. * indicates a *p*-value < 0.05 (paired *t*-test, *N* = 13). † indicates a significant difference between the percentage of experiments with a decreased and increased aggregation time related to the application of the linseed oil (*p* < 0.05, *z*-test, *N* = 13).</figcaption></figure>

The average amount of time that the **target females** spent in groups with members of the same sex did not significantly differ before and after the application of the linseed oil (0.2 ± 6.2 min, Figure 2C, *p* = 0.962, paired *t*-test, *N* = 13). In six trials out of 13, the target females spent on average 5.0 ± 3.5 min longer in groups after oil treatment (blue vertical stripes in Figure 2D). However, the proportion of trials exhibiting an increased and decreased aggregation time did not differ significantly (Figure 2D, *p* = 0.687, *z*-test, *N* = 13). Egg pods laid by females were often found in the isolation boxes and one time inside the arena after the trial. The average distance covered by the target females did not change after linseed oil application (Table S1, *p* = 0.814, paired *t*-test, *N* = 13). Observations performed after linseed oil application revealed that, in 69.2% of trials performed with females, six females, including the target one, were active, behaved normally and moved towards each other. In the other 30.8%, the target females were less active and only walked along the borders of the arena.
### 3.3. Pooled Aggregation Time after Linseed Oil Treatment

In more than half of all trials performed with either males or females, the total amount of time that the target individual spent in groups of at least two individuals decreased by more than 10% after the application of linseed oil (Figure 3A,B). An increase in the amount of aggregation time, however, was found in only about one-third of the trials. Averaging over all trials and sexes, the time spent in an aggregation decreased by only 2.2 ± 6 min (8.7 ± 24.5% of the observation time), which indicates that the application of linseed oil did not significantly change the total aggregation time (*p* = 0.077, paired *t*-test, *N* = 26). However, in 53.8% of trials in which the target individuals spent less time in groups, the aggregation time was significantly reduced by 7 ± 2.9 min after the application of linseed oil (red striped segment in Figure 3B, *p* < 0.001, paired *t*-test, *N* = 14). In only 30.8% of trials, the target individuals significantly extended the average time spent in aggregation by 5 ± 3.1 min (blue-striped segment in Figure 3B, *p* = 0.008, Wilcoxon signed-rank test, *N* = 8) after treatment. In 15.4% of all trials, there was no change in the aggregation time of the target individuals of more than 10% (black segment in Figure 3B).

<figure><img src="image_114.png" /><figcaption>**Figure 3.** Time that the target individual spent in male and female groups. (A) Aggregation time of the target individual before and after treatment with linseed oil. Red lines: Time spent in groups decreased. Blue lines: Time spent in groups increased. Black lines: Time spent in groups did not change by more than 10%. (B) Percentage of experiments in which aggregation time decreased (red horizontal stripes), increased (blue vertical stripes) or was left unchanged (black area) after linseed oil treatment. Total observation time = 25 min, *N* = 26 experiments.</figcaption></figure>

### 3.4. Sham Operation

To exclude an influence caused by the handling of the target individual, we quantified the total amount of time the target locust spent in groups of at least two individuals before and after the wings were brushed with a clean brush (results are shown in Table S2). Averaging over all trials and sexes,
the time spent in an aggregation decreased by only 0.8 ± 6.2 min (2.7 ± 20.8% of the observation time), which shows that the clean brush did not change the total aggregation time significantly (*p* = 0.538, paired *t*-test, *N* = 24). The average amount of time target **males** spent in groups with members of the same sex was reduced by only 1.2 ± 6.2 min (3.9 ± 20.7% of the observation time) after sham operation. The average amount of time the target **females** spent in groups with members of the same sex in this experiment was reduced by only 0.4 ± 6.5 min (1.4 ± 21.7% of the observation time).

### 3.5. Group Exposure to Linseed Oil and Dead Bodies

The average amount of time males or females spent in the target corner did not change significantly after the addition of the filter paper containing linseed oil (Table 2, males: *p* = 0.460, paired *t*-test, *N* = 6; females: *p* = 0.451, paired *t*-test, *N* = 5).

<table><caption>**Table 2.** Time any individual spent in the target corner of the arena before and after the addition of the linseed oil. Males and females were tested in separate experiments. Total observation time = 25 min, Group size = 5 individuals, *N* = 11.</caption><thead><tr><th rowspan="2">Experiments</th><th colspan="2">Time Spent in the Target Corner of the Arena</th></tr><tr><th>Control</th><th>Linseed Oil</th></tr></thead><tbody><tr><td rowspan="5">Females</td><td>20.4</td><td>20.4</td></tr><tr><td>10.1</td><td>11.9</td></tr><tr><td>15.8</td><td>18.9</td></tr><tr><td>20.9</td><td>17.3</td></tr><tr><td>16.8</td><td>21.4</td></tr><tr><td>Average of females</td><td>16.8 ± 4.4</td><td>18.0 ± 3.7</td></tr><tr><td rowspan="6">Males</td><td>14.6</td><td>14.6</td></tr><tr><td>16.9</td><td>18.7</td></tr><tr><td>17.9</td><td>15.1</td></tr><tr><td>11.4</td><td>13.3</td></tr><tr><td>16.0</td><td>15.3</td></tr><tr><td>16.8</td><td>21.9</td></tr><tr><td>Average of males</td><td>15.6 ± 2.3</td><td>16.5 ± 3.2</td></tr><tr><td>Total average</td><td>16.1 ± 3.3</td><td>17.2 ± 3.4</td></tr></tbody></table>

The average amount of time males or females spent in the target corner did not change significantly after the addition of the dead bodies (Table 3, males: *p* = 0.894, paired *t*-test, *N* = 6; females: *p* = 0.381, paired *t*-test, *N* = 5).

<table><caption>**Table 3.** Time any individual spent in the target corner of the arena before and after the addition of the intact dead bodies. Males and females were tested in separate experiments. Total observation time = 25 min, Group size = 5 individuals, *N* = 11.</caption><thead><tr><th rowspan="2">Experiments</th><th colspan="2">Time Spent in the Target Corner of the Arena</th></tr><tr><th>Control</th><th>Dead Bodies</th></tr></thead><tbody><tr><td rowspan="5">Females</td><td>7.5</td><td>23.8</td></tr><tr><td>18.9</td><td>21.3</td></tr><tr><td>13.8</td><td>11.8</td></tr><tr><td>12.1</td><td>20.6</td></tr><tr><td>14.8</td><td>8.9</td></tr><tr><td>Average of females</td><td>13.4 ± 4.2</td><td>17.3 ± 6.5</td></tr></tbody></table>
<table><caption>**Table 3.** *Cont.*</caption><thead><tr><th rowspan="2">Experiments</th><th colspan="2">Time Spent in the Target Corner of the Arena</th></tr><tr><th>Control</th><th>Dead Bodies</th></tr></thead><tbody><tr><td rowspan="6">Males</td><td>11.2</td><td>6.7</td></tr><tr><td>8.2</td><td>14.2</td></tr><tr><td>14.8</td><td>14.4</td></tr><tr><td>18.3</td><td>17.9</td></tr><tr><td>19.0</td><td>20.8</td></tr><tr><td>9.6</td><td>8.3</td></tr><tr><td>Average of males</td><td>13.5 ± 4.6</td><td>13.7 ± 5.4</td></tr><tr><td>Total average</td><td>13.5 ± 4.2</td><td>15.3 ± 5.9</td></tr></tbody></table>

### 3.6. *Responses of Males Towards Crushed Bodies*

In an observation period of 25 min, the average amount of time **males** spent in the target corner was significantly decreased by 6.4 ± 4.9 min (Table 4, *p* = 0.003, paired *t*-test, *N* = 10) after the addition of the crushed male bodies.

<table><caption>**Table 4.** Time any individual spent in the target corner of the arena before and after the addition of the crushed male bodies. Total observation time = 25 min, Group size = 5 individuals, *N* = 10.</caption><thead><tr><th rowspan="2">Experiments</th><th colspan="2">Time Spent in the Target Corner of the Arena</th></tr><tr><th>Control</th><th>Crushed Males</th></tr></thead><tbody><tr><td rowspan="10">Males</td><td>11.6</td><td>9.3</td></tr><tr><td>18.4</td><td>18.6</td></tr><tr><td>15.7</td><td>2.1</td></tr><tr><td>14.9</td><td>15.1</td></tr><tr><td>20.9</td><td>12.9</td></tr><tr><td>21.0</td><td>16.9</td></tr><tr><td>21.2</td><td>12.8</td></tr><tr><td>13.3</td><td>6.6</td></tr><tr><td>16.3</td><td>8.1</td></tr><tr><td>23.2</td><td>10.3</td></tr><tr><td>Average</td><td>17.7 ± 3.9</td><td>11.3 ± 5.0</td></tr></tbody></table>

## 4. Discussion

After isolating individuals for three days, we observed a general tendency for these individuals to form groups during the observation period (see Table 1), which is in contrast to solitary-reared locusts that are less active and freeze when they sense movement in the arena [5,10]. Furthermore, the amount of time any group member spent in groups was not influenced by brushing the wings of the target individuals with linseed oil (Table 1). Brushing the wings of the target locusts with linseed oil, however, affected the amount of time these individuals spent in proximity to other individuals in most trials (see Figure 3). In contrast, brushing the wings with a clean brush in the sham operation experiment did not change the average group formation time significantly (see Table S2). This suggests that the reduced group formation time observed in males is caused by linseed oil and less likely by physical stimulation. Brushing the wings of the target individuals with linseed oil did not change the average distance covered by the target locusts (see Table S1), which suggests that the reduced group formation time observed in males is not caused by an overall reduction in the activity of the target locust. Linseed oil is rich in unsaturated fatty acids, and some of these acids (especially oleic and linoleic acid) have been described as necromones that are released by injured and dead insects. These necromones are known to trigger distinct behaviour patterns in other individuals of the same or different species [14–16]. Therefore, we tested whether linseed oil could be used as an agent to
control the aggregation behaviour of gregarious locusts by investigating the aggregation behaviour of
members of both sexes in controlled experiments.

A reduced group formation time of males after the linseed oil application (see Figure 2A,B) may be the result of necrophobic avoidance behaviour, which may be absent in females most likely because of oviposition pheromone release. Males in this study showed a necrophobic avoidance and tended to move away from the target individual that released “the smell of death” in order to avoid possible infections that can be transmitted by swarm mates or indicates an injury from a predator attack [14]. This aversion to necromone fatty acids (especially oleic and linoleic acids) were reported in a large number of invertebrates [19]. However, locusts are also known to cannibalise other swarm mates in situations where food is scarce. We have not observed cannibalistic tendencies in our study because all individuals were fed with fresh grass during the isolation period. An interesting observation made in the course of this study was that the target individuals were no longer involved in mounting behaviours after being brushed with linseed oil. This male–male mounting behaviour is rather common in the absence of females, and our results indicate that the observed change in male behaviour was mediated by linseed oil. This result differs from that of Clancy et al. [20], who observed an increase in the frequency of male–male mounting (MMM) behaviour in desert locust males suffering from *Metarhizium acridum*, a fungal infection. This reduction in MMM is similar to the effect caused by “phenylacetonitrile” (PAN, also known as benzyl cyanide), which is a mature male volatile that prevents males from being mounted (homosexuality) by other males [5,21]. It is mainly released from the wings and the hind legs [22] and acts as a strong repellent for mature males to hide a female from other competing males (a courtship inhibition pheromone) [5,21]. Brushing the wings with linseed oil likely dilutes the concentration of PAN on the wings of the target male due to the high hydrophobicity of this pheromone. Therefore, we conclude that the repellent effect related to our treatment is mediated by fatty acids but less likely by PAN. However, Bashir et al. [23] proved that PAN has a solitarising effect (anti-gregarisation) on the hopper bands, and another recent study revealed that PAN serves as an antipredator defence in gregarious migratory locusts as it is converted into a hypertoxic cyanide (HCN) when they are under attack [24]. Since mature males exclusively produce aggregation pheromones that are attractive to members of both sexes [25–27], a reduction in male group formation caused by linseed oil may have important consequences for the formation and persistence of locust swarms.

We observed no significant difference in the average amount of time the target females spent in
groups before and after the application of linseed oil (see Figure 2C,D). In almost half of all trials,
there was a significant increase in the average amount of aggregation time by 5 ± 0.6 min. This result can
be attributed to the attraction between female individuals mediated by the oviposition pheromone [25].
Since the target females were treated after the control recording ended, this might lead to an increase in
the amount of oviposition pheromone released in the arena with time. This consequently increases the
time the females spent in groups even in the presence of fatty acids necromones. This female–female
attraction ensures the spatial aggregation of egg pods, which increases the survival rates and supports
gregarious cohesion among members of the next generation. Furthermore, it has been also shown that
ovipositing females of desert locusts aggregate responding to a pheromone produced by alive or dead
individuals in all development stages [28]. In 30.8% of trials performed with females in this study,
brushed target females stayed away, were less active and only walked on the borders of the arena.
This may indicate that females carrying the smell of death seem to avoid being eaten by conspecifics
because it is known that desert locusts show cannibalistic behaviour beginning in the fifth-instar stage,
and especially adults may be regarded as an important source of proteins by conspecifics [29,30].
Bazazi et al. [31] studied collective motion and cannibalism in marching bands of desert locust nymphs
and was able to show that individuals that were injured by conspecifics may suffer an increased risk
of cannibalism. Since females have a higher demand for the proteins involved in egg development,
they may respond to the linseed-oil-treated target individual in a different way than males.

To study possible attraction/avoidance behaviour of the desert locusts to necromones, we tested
the time males and females spent in the presence of a stationary linseed oil target and dead bodies.
While we found no difference in the average amount of time males and females spent in the target corner after the addition of the linseed oil or dead bodies (see Tables 2 and 3), there was a significant reduction in this parameter in the experiment performed with crushed male bodies as males spent less time next to them (see Table 4). A response to freshly dead individuals is rather unlikely since the responses to fatty acid necromones increased over time [14,19,32]. According to our results, responses of some genera of isopods were very weak to intact dead bodies (disease) compared to crushed bodies or body extracts [14]. The latter indicates injury resulting from predation and swarm mates should avoid them.

Sex-specific responses to fatty acid necromones were also described in the cricket *Acheta domesticus*, where females respond to body extracts less than males, as females might be less risk averse because they seek out singing males and explore oviposition sites [19]. Another study investigated the repellent effect of various fatty acid necromones on cockroach males and females. In both sexes, the percentage of repellence was strictly dose dependent and the percentage of oleic acid repellence was more significant in cockroach males (70%) than females (43%) [33]. The results obtained from males in our study indicate that fatty acids from linseed oil may change the aggregation behaviour of desert locusts. A similar solitarising effect has been found in *S. gregaria* when nymphs were exposed to faeces of crowded locusts [34].

**5. Conclusions**

A novel botanical pesticide formulation uses linseed oil as the main component and was found to be highly effective against gregarious desert locusts as well as migratory locusts [11]. Since linseed oil contains unsaturated fatty acids that have been shown to act as necromone cues in different insect species, it was of interest to determine whether this oil affects group formation in gregarious desert locusts. Brushing the wings of single gregarious desert locust males with this oil significantly decreased the amount of aggregation time they spent in male groups in the majority of the trials (Figure 2). In contrast, most treated females either did not display alterations in aggregation time or the aggregation time increased (Figure 2). These sex differences in aggregation behaviour may be explained by the release of different pheromones produced by adult males and females in the gregarious phase. Since a reduction in the amount of aggregation time among males leads to a reduction in tactile stimuli that have been shown to be highly gregarizing [8,9], linseed oil seems to be a promising candidate agent for the control of aggregation behaviour in gregarious desert locusts, as it may even disrupt swarm formation once a certain percentage of individuals have come into contact with linseed oil in the course of botanical pesticide treatments. Therefore, fatty acids that act as necromones and are contained in linseed oil should be considered in pest management as previously suggested by Yao et al. [14]. Future studies need to reveal the origin of this behaviour by exposing males and females to different concentrations and types of necromones in controlled laboratory conditions.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4395/10/10/1458/s1, Table S1: Distance covered by the target individual before and after the application of linseed oil. Males and females were tested in separate experiments. Total observation time = 25 min, Group size = 6 individuals, N = 26. Table S2: Time the target individual spent in groups before and after sham operation. Males and females were tested in separate experiments. Total observation time = 25 min, Group size = 6 individuals, N = 14.

**Author Contributions:** Z.A.S.A. and M.H. conceived and designed the research. Z.A.S.A. conducted the experiments. M.H. supervised the experiments. Z.A.S.A. analysed the data and drafted the manuscript. Both authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Egyptian Ministry of Higher Education, Cultural Affairs and Mission Sector. Open Access Funding by the University of Graz.

**Acknowledgments:** Many thanks to Boris P. Chagnaud for his critical reading and valuable comments.

**Conflicts of Interest:** The authors declare no competing interests.

**Data Availability:** Data will be made accessible upon request.
**Compliance with ethical standards:** The experimental procedures were performed at the Institute of Biology (University of Graz). All experiments comply with the current Austrian and European Community laws for the ethical treatment of animals and are in line with the ASAB Guidelines for the Use of Animals in Research.

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<figure><img src="image_115.png" /></figure>

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Article

# Fungal-Based Biopesticide Formulations to Control Nymphs and Adults of the Desert Locust, *Schistocerca gregaria* Forskål (Orthoptera: Acrididae): A Laboratory and Field Cage Study

Waqas Wakil <fnref n="1" />,<fnref n="2" />,* ID, Muhammad Usman Ghazanfar <fnref n="3" />, Muhammad Usman <fnref n="1" /> ID, David Hunter <fnref n="4" /> ID
and Wangpeng Shi <fnref n="5" />,* ID

**Abstract:** This is the first field study in which we have tested the efficacy of four different entomopathogenic fungal (EPF) formulations together in single study—i.e., Green Muscle, Green Guard, *Metarhizium anisopliae*, and an isolate of *Beauveria bassiana* (isolate WG-11)—against nymphs and adults of the desert locust, *Schistocerca gregaria* Forskål (Orthoptera: Acrididae). We conducted several different studies: (a) lethal bioassay against the 3rd, 5th, and adult stages under laboratory conditions; (b) sublethal effects on the reproduction, diet consumption, fecal production, and weight gain; (c) a greenhouse trial; and (d) a field cage trial. Under laboratory conditions, all EPF formulations caused significant mortality, and the highest efficacy was observed with Green Muscle, followed by Green Guard, *B. bassiana*, and *M. anisopliae*. Susceptibility was found to be greatest in 3rd-instar nymphs, followed by 5th instars, and then adults. Along with lethal effects, sublethal doses of EPF reduced the number of egg pods per female, total eggs per pod, and egg hatching, while extending nymphal developmental time and reducing adult longevity; again, Green Muscle performed better. Sublethal doses not only retarded reproduction, but also caused behavioral changes, including reductions in food consumption, fecal production, and weight gain. All EPF formulations not only produced significant mortality in laboratory conditions, but also performed very well under the greenhouse and field conditions. The maximum mortality against 3rd-instar (81.7% and 74.0%), 5th-instar (73.3% and 65.1%), and adult locusts (67.5% and 58.9%) was observed when using Green Muscle under greenhouse and field trials, respectively. The current study showed that all of the EPF formulations have the potential to reduce pest populations, and could be used in the integrated pest management program.

**Keywords:** desert locust; *Metarhizium acridum*; *Metarhizium anisopliae*; *Beauveria bassiana*; lethal effect; sublethal effect; greenhouse; field efficacy

**Citation:** Wakil, W.; Ghazanfar, M.U.; Usman, M.; Hunter, D.; Shi, W. Fungal-Based Biopesticide Formulations to Control Nymphs and Adults of the Desert Locust, *Schistocerca gregaria* Forskål (Orthoptera: Acrididae): A Laboratory and Field Cage Study. *Agronomy* 2022, 12, 1160. https://doi.org/10.3390/agronomy12051160

Academic Editor: Beatriz Gámiz

Received: 22 July 2021

Accepted: 11 August 2021

Published: 11 May 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

<figure><img src="image_116.png" /></figure>

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

## 1. Introduction

Locust plagues are considered a major constraint in sustainable agriculture production [1–3]. The desert locust, *Schistocerca gregaria* Forskål (Orthoptera: Acrididae), has produced extensive losses of different food crops in Africa and Asia [4–6], especially during locust upsurges. In 2019, *S. gregaria* entered the province of Baluchistan in Pakistan from Iran, and then spread to different parts of Sindh, and finally to all four provinces. It was estimated that Pakistan faced a loss of PKR 353 billion and 464 billion if 25% damage occurred to Rabi (sown in winter) and Kharif crops (sown in spring), respectively

<footnote n="1">¹ Department of Entomology, University of Agriculture, Faisalabad 38040, Pakistan; usmanbhattiuaf@gmail.com</footnote>

<footnote n="2">² Senckenberg German Entomological Institute, D-15374 Müncheberg, Germany</footnote>

<footnote n="3">³ Department of Plant Pathology, College of Agriculture, Sargodha University, Sargodha 40100, Pakistan; usmanghazanfar1972@gmail.com</footnote>

<footnote n="4">⁴ Locust and Grasshopper Control, McKellar, ACT 2617, Australia; davidmhunter100@gmail.com</footnote>

<footnote n="5">⁵ Department of Entomology, College of Plant Protection, China Agricultural University, Beijing 100083, China</footnote>

<footnote>* Correspondence: waqaswakeel@hotmail.com (W.W.); wpshi@cau.edu.cn (W.S.)</footnote>
(https://www.dawn.com/news/1555487; accessed on 12 April 2021). There was apprehension that, if left uncontrolled, the number of locusts would increase 500-fold by June 2020 in East Africa [7]—potentially the worst infestation in past 100 years [8]. Climate change aggravates this situation due to high temperatures and increased precipitation, which accelerate locust growth and reproduction [9]. *Schistocerca gregaria* has three different developmental stages: egg, nymph, and adult. Adult females lay eggs in the form of egg pods in the soil at 5–10-cm depth [10]. Egg pods are 3–4 cm long, white in color, and shaped like bananas. Egg hatching occurs in 10–14 days when it is hot, or up to 65 days when it is cool, with each egg pod consisting of 80–160 eggs [11]. Hatched eggs produce nymphs known as hoppers that have 5–6 instars, and the developmental period from the first to the last instar is 28–48 days, followed by the adult stage, which can last for several months [12,13].

Management of locust plagues depends heavily on the use of chemical insecticides [14,15]. Prior to 1980, the organochlorine insecticide dieldrin was effectively used for many years to control this plague because of its persistence. However, due to their hazardous effects on animals and the environment [4], the use of organochlorines is now banned, and locust control mainly relies on organophosphate insecticides [16] that have low persistence, making them less effective than dieldrin [17]. Between 2003 and 2005, to combat the locust outbreak, over 13 million hectares were treated using 13 million liters of chemical insecticides [18]. During the 2019–20 locust outbreak in Pakistan, the Department of Plant Protection (DPP) sprayed 150,839 L of pesticides over an area of 300,595 hectares to suppress the locust population. The effects of non-judicious, intensive, and heavy use of chemical pesticides are probably underestimated, leading to environmental problems resulting from lethal effects on the whole ecosystem [19,20].

Knowing the hazardous effects of conventional insecticides, the development of safe alternatives is essential [21,22]. The utilization of natural enemies such as insect predators and parasitoids as control methods is not likely to be effective, due to the rapid growth and highly dispersed movement of locusts [4,15]. Green technology such as biological control uses microorganisms and birds to help to reduce the locust population [9], reducing the toxic impact of pesticides on the ecosystem. Microbial biopesticides could be an effective alternative to synthetic insecticides in locust management [6,23,24]. Microbial-based control methods, including the utilization of entomopathogenic fungi (EPF), have an extra benefit in that they invade the insect host via direct contact with the insect's integument instead of ingestion of the pathogen [23,25]. The Food and Agriculture Organization (FAO) has been working for the past 20 years in different countries on the development of biopesticides based on *Metarhizium acridum* (Driver and Milner) J.F. Bisch, Rehner and Humber (Hypocreales: Clavicipitaceae) to infect grasshoppers and locusts [18]. In field conditions, locusts can be exposed to *M. acridum* conidia in three ways: direct exposure to spray application, secondary pick-up of conidia from treated vegetation, or horizontal transmission from infected individuals during copulation and during aggregation phenomena [26]. While, there have been a number of studies on the efficacy of EPF such as *M. acridum* [15,26–30] and *Beauveria bassiana* (Bals.-Criv.) Vuill. (Hypocreales: Cordycipitaceae) [29,31] against *S. gregaria*, ours is the first to test four different formulations against *S. gregaria* in a single study.

In addition to lethal effects, EPF have sublethal effects that can disturb overall pest status [27,32,33]. Reduction in feeding was observed among acridid insect pests following exposure to sublethal doses of entomopathogens [32,34–38]. Reduced fecundity was observed in surviving adults after being exposed to sublethal doses [27,39,40]. Similar effects have also been observed in other insect pests, such as houseflies and mosquitoes [41–43]. This is the first study to test the effects of different EPF on the survival and longevity of different developmental stages of *S. gregaria*.

Many insect pests are able to thermoregulate their body temperature across different environmental conditions [44]. Acridid insects not only change their body temperature with respect to location, but also alter their body temperature (behavioral fevers) when infected with fungal conidia under field conditions. They assume basking postures or
locations that elevate their body temperature above optimal temperatures for microbial
growth [45,46]. Commonly, lower mortality was observed in the field conditions compared
to the laboratory [47]. Consequently, greenhouse and field cage experiments were included
in this study in order to determine whether locusts exposed to sunlight could thermoregu-
late and reduce EPF efficacy compared to the laboratory. The objective of this study was
to evaluate the efficacy of two different commercial formulations of *M. acridum*—one of
*M. anisopliae*, and one local strain of *B. bassiana* (isolate WG-11)—against 3rd- and 5th-instar
nymphs and adults of *S. gregaria* in laboratory, greenhouse, and field trials.

**2. Materials and Methods**

2.1. *Insect Collection and Rearing*

Adults of *S. gregaria* were collected from a swarm in Bhakkar Punjab (Pakistan) and reared in the laboratory inside wooden cages (30 cm × 30 cm × 30 cm). The cages were made with mesh wires on three sides of cages and on the roof. The front side of each cage was fitted with a door that contained white cloth with sleeves for operations like insect introduction, diet supplementation, and cleaning. The bottom of the cage consisted of several small holes (10-cm diameter) for egg laying. Plastic cups filled with sand with appropriate moisture content were placed below the wooden cage for egg collection. The adults were provided with wheat seedlings and wheat bran as food. Three light bulbs (60 W) were placed on the top of the roof for heating, and were switched on for 6–8 h each day for thermoregulation, which enabled basking behavior, as would occur in the field. After egg laying, the small plastic cups (covered with mosquito wire mesh) were placed under the light bulb (60 W) for 6–8 h to accelerate embryonic development. Upon hatching, first-instar nymphs were released inside the cage for subsequent development [26].

2.2. *Entomopathogenic Fungi and Their Culturing*

The biopesticides used in this study included two commercial products based on *M. acridum*—Green Guard (BASF, CSIRO FI (=ARSEF 324)) and Green Muscle (Elephant Vert, IMI 330189 (=ARSEF7486))—one *Metarhizium anisopliae* (Metchnikoff) Sorokin (Hypocreales: Clavicipitaceae) (CQMCC No. 0877) from China, and a local strain of *B. bassiana* (isolate WG-11) originally isolated from soil samples collected from Lal Suhanra, Punjab, (Pakistan). Each fungal isolate was individually inoculated on a potato dextrose agar (PDA) Petri plates (100 × 20 mm), wrapped with Parafilm, and finally placed in an incubator at 25 °C with a 14:10 h (light:dark) photoperiod. After 7–10 days, fungi were harvested using a sterile scalpel, followed by the conidial suspension being placed inside a falcon tube (50 mL) containing 30 mL of 0.05% Silwet L-77 solution. Eight glass beads were added to the tube and the solution was vortexed for about 5 min for proper agitation, and the desired conidial doses were determined using hemocytometer. Conidial viability of each fungal isolate was determined by spreading 0.1 mL of solution at rate of 1 × 10⁶ conidia/mL on Sabouraud dextrose agar with 1% yeast (SDAY) plates (two plates for each isolate) wrapped with Parafilm and placed in the incubator at 25 °C with a 14:10 h (light:dark) photoperiod for 16 h [48]. A coverslip was placed on each SDAY plate, and approximately 200 conidia were assessed for germination for one count, with a conidium considered to be germinated if the germ tube was longer than the conidia [48]. Two counts were taken from each plate, totaling four counts for each isolate [49,50].

2.3. *Bioassay against 3rd- and 5th-Instar Nymphs and Adults of S. gregaria*

The bioassays were conducted to assess the effect of different doses (1 × 10⁵, 1 × 10⁶,
1 × 10⁷, and 1 × 10⁸ conidia/mL) of entomopathogenic fungi against 3rd- and 5th-instar
nymphs and adults (two weeks after last molt) of *S. gregaria* at the Department of Entomol-
ogy, University of Agriculture Faisalabad, Punjab (Pakistan). Different doses of each fungal
isolate were prepared in 0.05% Silwet L-77 solution. Both nymphal instars and adults were
treated with a 1-mL suspension of entomopathogenic fungi using a 35-mL glass atom-
izer [51]. Control individuals were treated in the same fashion, but with 1 mL of 0.05% Sil-
wet L-77. After application, individuals were transferred to (30 cm × 30 cm × 30 cm) cages and provided with wheat bran and wheat seedlings grown in pots [16]. Each dose of specific formulations/isolates represents a treatment, each treatment consisted of three replicates (total 45 individuals, 15/replication), and the experiment was conducted twice (using new individuals and new materials), giving a total of six replicates [16,28]. Mortality was determined at 24-h intervals, with the final count at 12 days post-application [52]. The environmental conditions were maintained at a 14:10 h (light:dark) photoperiod, 30 °C, and 60% relative humidity (RH) [51].

## 2.4. *Effect of a Sublethal Dose on the Reproduction and Development of S. gregaria*

The effect of a sublethal dose (1 × 10⁴ conidia/mL) of each fungal isolate was examined against adults of *S. gregaria*. For this, a group of 30 adults (15 females and 15 males) was treated as described in the previous section. The cages were provided with 25-cm-diameter plastic cups filled with clean sand, and were provided with appropriate moisture to encourage egg laying. The total number of egg pods laid by each female, average number of eggs present per pod, and percentage of eggs hatching was determined. The egg pods were collected at the end of experiment from among the different treatments, and mean egg pods per female were determined using the number of females still alive on the day of first oviposition. The egg pods were dissected and the total number of eggs per pod assessed. Then, eggs were kept under optimal conditions of 27 °C, 60% RH, and total egg hatching was determined among the different treatments [53]. After egg hatching, a group of 30 first-instar nymphs was collected from each replicate and kept inside the cages (30 cm × 30 cm × 30 cm) and provided with fresh diet (wheat seedlings and wheat bran). The developmental duration of different stages (1st-, 2nd-, 3rd-, 4th-, and 5th-instar nymphs, and adults) was determined. Each treatment consisted of three replicates, and the whole experiment was repeated twice, with new individuals and materials for each repetition.

## 2.5. *Effects of a Sublethal Dose on Diet Consumption, Weight Gain, and Frass Production of S. gregaria*

The effects of a sublethal dosage (1 × 10⁴ conidia/mL) on diet consumption, weight gain, and frass production of *S. gregaria* were determined. Fifth-instar nymphs were treated with a sublethal dose of EPF in a similar manner as in the above-mentioned bioassays. Individual 5th-instar nymphs were weighed and then transferred into a small plastic box and provided with a wheat seedling for feeding. Prior to feeding, diet weight was measured on weight balance and then transferred inside the small boxes for feeding. Daily unused diet was taken from the boxes and measured on the weight balance, and diet consumed was calculated by subtracting the remaining diet from the initial diet. During days 1–15 after inoculation, fecal pellets were collected daily from each cage (replicate). The fecal production per cage was oven-dried at 80 °C until constant weight (24 h) and then weighed. The fecal production per insect per day for each replicate was calculated by dividing the daily fecal production by the number of insects remaining alive on that date [34]. Insects were also weighed each day. Each treatment consisted of three replicates, with each replication containing 15 individuals (total 45 individuals per treatment), and the experiment was repeated twice, giving a total of six replicates.

## 2.6. *Greenhouse Trial*

The greenhouse experiment was conducted using nymphs (3rd- and 5th-instar) and adults of *S. gregaria*. The 3rd- and 5th-instar nymphs and adults were inoculated in a similar manner as in the laboratory bioassay, with 1 × 10⁹ conidia/mL. After inoculation, each individual of each specific stage was released inside a plastic pot (12.5 cm H × 8 cm D) that contained wheat seedlings and covered with a cylindrical metal cage (30 cm H × 8 cm D). All pots with cages were kept inside trays provided with water and placed on the bench top at 1-m height. Each treatment consisted of three replicates, with 20 pots per replicate (for each stage), and each pot contained one individual of a specific stage. The experiment
was repeated twice, giving a total of six replicates. Adult mortality was observed daily for 15 days [52]. In order to avoid scavenging by live locusts, their frass and dead were removed from the cages daily. Conditions were maintained at 30 °C, 16:8 h (light:dark) photoperiod, and 60% RH.

## 2.7. Field Trial

The effectiveness of different fungal isolates against 3rd- and 5th-instar nymphs and adults of *S. gregaria* was evaluated in the field cages (70 cm × 70 cm × 70 cm). These wooden cages with a door on the front were placed in the field, and 120 individuals of each stage were placed in each cage [51]. Each wooden cage served as replicate, and each treatment consisted of three replicates. As is common for field applications for Green Muscle [18,26], conidia were mixed with diesel and applied at a dose of 1 × 10⁹ conidia/mL using a knapsack sprayer. The experiment consisted of five treatments, with three replicates (three cages) in each treatment. The whole experiment was repeated twice [26], with new individuals and materials each time. The data on the locust mortality were taken daily by removing the dead from each field cage. Untreated wheat seedlings were provided in the cages in the late afternoon so that the locusts had fresh food all night. Sometimes there was mortality in the first two days due to transport and handling; to compensate for this, any mortality that occurred in the first two days, in both the treated and untreated cages, was ignored. The experiment endpoint was day 15 after the introduction of insects and treatment application.

## 2.8. Statistical Analysis

In the laboratory trials, the mortality among the different developmental stages (3rd- and 5th-instar nymphs, and adults) was corrected with control mortality using Abbott's formula [54]. The values were subjected to two-way analysis of variance (ANOVA), and treatment means were separated by Tukey's honestly significant difference (HSD) test [55]. The mortality among different stages and intervals was found to be less than 5%. The effects on reproduction, diet consumption, frass production, and weight gain were subjected to analysis of variance (ANOVA). The mortality in greenhouse and field trials was subjected to analysis of variance (ANOVA), and treatment means were separated by Tukey's HSD test. All of the statistical analysis was conducted using Minitab 17 software [56].

# 3. Results

## 3.1. Bioassay against 3rd- and 5th-Instar Nymphs and Adults of *S. gregaria*

Against 3rd-instar nymphs, significant differences were observed between different EPF within each dose at 5 ($F_{3,23} \le 6.05$; *p* < 0.01), 7 ($F_{3,23} \le 7.92$; *p* < 0.01), and 12 ($F_{3,23} \le 7.31$; *p* < 0.01) days post-treatment. Within each EPF, a significant dose response was observed ($F_{3,23} \le 13.6$; *p* < 0.01 at 5 days; $F_{3,23} \le 14.3$; *p* < 0.01 at 7 days; $F_{3,23} \le 17.2$; *p* < 0.01 at 12 days) post-treatment. The mortality of the EPF at the 10⁶ dose at 12 days ranged from 46.5 to 67.5%, while the 10⁸ dose resulted in 65–95% mortality. The dose response was such that increasing the dose 100-fold (from 10⁶ to 10⁸) meant mortality was reached 5 days earlier. Green Muscle produced mortality of 68% at the 10⁶ dose after 12 days, while similar mortality was observed at day 7 for the 10⁸ dose (Figure 1a–c). For *M. anisopliae*, there was 47% mortality at 12 days at the 10⁶ dose, and at 7 days at the 10⁸ dose (Figure 1b,c). The time taken to reach > 50% mortality was 5 days after a 10⁸ dose of Green Muscle, 7 days after a 10⁷ dose of Green Guard, 7 days after a 10⁸ dose of *B. bassiana*, and 12 days after a 10⁷ dose of *M. anisopliae* was applied. By the end of the experiment on day 12, maximum mortality was observed with Green Muscle, followed by *B. bassiana*, and *M. anisopliae*. Overall, mortality was greatest with Green Muscle and least with *M. anisopliae* at all time intervals and at all doses (Figure 1a–c).
<figure><img src="image_117.png" /><figcaption>**Figure 1.** Mean mortality percentages (±SE) of 3rd-instar nymphs of *S. gregaria* after (a) 5 days, (b) 7 days, and (c) 12 days of treatment with various doses (10⁵, 10⁶, 10⁷, and 10⁸ conidia/mL) of Green Muscle, Green Guard, *B. bassiana*, or *M. anisopliae*. Mortality data were corrected for control mortality, which was <5%. For each subfigure: different lowercase letters indicate significant differences in mortality between each of the EPF; different uppercase letters indicate significant differences in mortality between doses (Tukey's HSD test at *p* ≤ 0.05).</figcaption></figure>

Clear treatment effects were observed against 5th-instar nymphs at different dose rates at 5 ($F_{3,23} \le 15.1$; *p* < 0.01), 7 ($F_{3,23} \le 22.4$; *p* < 0.01), and 12 ($F_{3,23} \le 17.0$; *p* < 0.01) days post-treatment. All of the EPFs had a significant dose response ($F_{3,23} \le 4.28$; *p* < 0.01 at 5 days; $F_{3,23} \le 5.92$; *p* < 0.01 at 7 days; $F_{3,23} \le 3.88$; *p* < 0.01 at 12 days). The dose response was such that increasing the dose 100-fold (from $10^6$ to $10^8$) meant mortality was reached just under 5 days earlier. The mortality due to the EPF at the $10^6$ dose at 12 days ranged from 37.1 to 54.8%. For Green Muscle; there was 55% mortality at 12 days at a dose of $10^6$,
similar to the 61% mortality at 7 days at a dose of 10⁸ (Figure 2b,c). Until day 5, no EPF was able to kill 50% of 5th-instar nymphs, while at 7 days of exposure only two EPF (Green Muscle and Green Guard) caused > 50% mortality at a level not statistically different from one another, with the greatest mortality from Green Muscle (Figure 2a–c).

<figure><img src="image_118.png" /><figcaption>(b)</figcaption></figure>

<figure><img src="image_119.png" /><figcaption>(c)</figcaption></figure>

<figure><img src="image_120.png" /></figure>

**Figure 2.** Mean mortality percentages (±SE) of 5th-instar nymphs of *S. gregaria* after (a) 5 days, (b) 7 days, and (c) 12 days of treatment with various doses (10⁵, 10⁶, 10⁷, and 10⁸ conidia/mL) of Green Muscle, Green Guard, *B. bassiana*, or *M. anisopliae*. Mortality data were corrected for control mortality, which was <5%. For each subfigure: different lowercase letters indicate significant differences in mortality between each of the EPF; different uppercase letters indicate significant differences in mortality between doses (Tukey's HSD test at *p* ≤ 0.05).

Against adults, each dose rate of EPF produced significantly more mortality compared to the control treatment ($F_{3,23} \le 7.23$; *p* < 0.01), ($F_{3,23} \le 5.53$; *p* < 0.01), and ($F_{3,23} \le 3.17$; *p* < 0.01) at 5, 7, and 12 days after treatment, respectively. A clear dose response in terms of mortality was observed in different EPF at ($F_{3,23} \le 16.6$; *p* < 0.01) 5 days, ($F_{3,23} \le 17.1$; *p* < 0.01) 7 days, and ($F_{3,23} \le 20.6$; *p* < 0.01) 12 days post-application. Once again, the dose response was such that increasing the dose 100-fold (from $10^6$ to $10^8$) resulted in mortality being reached just under 5 days earlier. The mortality of the EPF at the $10^6$ dose at 12 days ranged from 49% for Green Muscle to 34% for *M. anisopliae*—slightly less than the levels
reached at 7 days with a 10⁸ dose. No EPF caused 50% mortality at 5 days, and even at 7 days, only two EPF caused 50% mortality, and even then only at the highest conidial dose. After 12 days, all EPF caused > 50% mortality at the two highest conidial doses. As with treatments of 3rd- and 5th-instar nymphs, the greatest mortality was observed with Green Muscle, followed by Green Guard, *B. bassiana*, and *M. anisopliae* (Figure 3a–c).

<figure><img src="image_121.png" /><figcaption>(b)</figcaption></figure>

<figure><img src="image_122.png" /><figcaption>(c)</figcaption></figure>

<figure><img src="image_123.png" /></figure>

**Figure 3.** Mean mortality percentages (±SE) of *S. gregaria* adults after (a) 5 days, (b) 7 days, and (c) 12 days of treatment with various doses (10⁵, 10⁶, 10⁷, and 10⁸ conidia/mL) of Green Muscle, Green Guard, *B. bassiana*, or *M. anisopliae*. Mortality data were corrected for control mortality, which was <5%. For each subfigure: different lowercase letters indicate significant differences in mortality between each of the EPF; different uppercase letters indicate significant differences in mortality between doses (Tukey's HSD test at *p* ≤ 0.05).
Overall, 3rd-instar nymphs were found to be more susceptible than 5th-instar nymphs or adults.

## 3.2. Effects of Sublethal Doses on the Reproduction and Development of S. gregaria

Sublethal doses of EPF seriously affected the reproduction capability of *S. gregaria*, and also retarded the growth of their offspring. The females treated with all EPF except *M. anisopliae* produced fewer egg pods compared to the untreated controls ($F_{4,29} = 28.1$; $p < 0.01$), with no significant difference between the Green Muscle and Green Guard treatments (Table 1). In addition, there were fewer eggs per pod with EPF treatments ($F_{4,29} = 73.1$; $p < 0.01$), with Green Muscle treatments leading to the lowest number of eggs per pod (Table 1). Of the eggs laid, hatching was lowest with the Green Muscle and Green Guard treatments, with both *B. bassiana* and *M. anisopliae* resulting in intermediate levels between the other EPF and the controls. Even though the offspring themselves were uninfected, the offspring of infected females had an increase in nymphal developmental time ($F_{4,29} = 7.20$; $p < 0.01$) and a reduction in adult longevity compared to the untreated controls ($F_{4,29} = 41.2$; $p < 0.01$).

<table><caption>**Table 1.** Effects of applying one sublethal dose ($1 \times 10^4$ conidia/mL) of Green Muscle, Green Guard, *B. bassiana*, or *M. anisopliae* to *S. gregaria* adults on the number of egg pods/female, number of eggs/pod, egg hatching, development time for offspring of different instars, and survival of resulting adults. Data are means $\pm$ SE, and within each column and treatment, different letters indicate significant differences (Tukey's HSD test at $p \le 0.05$).</caption><thead><tr><th rowspan="2">Treatment</th><th rowspan="2">No. of Egg Pods/Female</th><th rowspan="2">Eggs/Pod</th><th rowspan="2">Egg Hatching (%)</th><th colspan="5">Development Time (days)</th></tr><tr><th>L1</th><th>L2</th><th>L3</th><th>L4</th><th>L5</th><th>Adult</th></tr></thead><tbody><tr><td>Green Muscle</td><td>0.76 ± 0.11 c</td><td>23.13 ± 1.75 d</td><td>34.44 ± 4.99 c</td><td>8.63 ± 0.32 a</td><td>7.31 ± 0.17 a</td><td>7.51 ± 0.20 a</td><td>7.90 ± 0.26 a</td><td>10.21 ± 0.42 a</td><td>18.65 ± 1.17 d</td></tr><tr><td>Green Guard</td><td>1.06 ± 0.12 bc</td><td>34.54 ± 1.54 c</td><td>45.33 ± 4.89 bc</td><td>7.96 ± 0.23 ab</td><td>6.86 ± 0.20 ab</td><td>6.96 ± 0.21 ab</td><td>7.48 ± 0.21 ab</td><td>9.64 ± 0.32 ab</td><td>27.30 ± 2.16 cd</td></tr><tr><td><i>B. bassiana</i></td><td>1.41 ± 0.11 b</td><td>40.58 ± 1.34 bc</td><td>53.33 ± 3.56 b</td><td>7.22 ± 0.17 bc</td><td>6.20 ± 0.17 bc</td><td>6.71 ± 0.17 b</td><td>7.11 ± 0.20 abc</td><td>9.25 ± 0.27 abc</td><td>33.20 ± 1.81 bc</td></tr><tr><td><i>M. anisopliae</i></td><td>2.01 ± 0.16 a</td><td>47.78 ± 2.29 b</td><td>61.11 ± 3.18 b</td><td>6.46 ± 0.19 cd</td><td>5.60 ± 0.16 cd</td><td>5.85 ± 0.15 c</td><td>6.76 ± 0.28 bc</td><td>8.41 ± 0.28 bc</td><td>38.71 ± 2.59 b</td></tr><tr><td>Control</td><td>2.56 ± 0.15 a</td><td>67.36 ± 2.44 a</td><td>85.55 ± 4.00 a</td><td>6.04 ± 0.16 d</td><td>5.08 ± 0.11 d</td><td>5.01 ± 0.13 d</td><td>6.20 ± 0.21 c</td><td>8.06 ± 0.28 c</td><td>55.95 ± 2.75 a</td></tr><tr><td><i>F</i></td><td>28.3</td><td>73.1</td><td>21.6</td><td>22.1</td><td>28.0</td><td>30.7</td><td>7.20</td><td>7.30</td><td>41.2</td></tr><tr><td><i>p</i></td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td></tr></tbody></table>

## 3.3. Effects of a Sublethal Dose on Diet Consumption, Weight Gain, and Frass Production of S. gregaria

Sublethal doses induced significant behavioral changes in fifth-instar *S. gregaria*, with lower diet consumption, frass production, and weight compared to controls; most effects were already seen at 3 days after infection, and were clearly evident by 6 days (Figure 4a-c). As the days progressed, diet consumption declined in the treated groups ($F_{4,29} \le 19.6$; $p < 0.01$), but increased in the untreated controls ($F_{4,29} \le 6.51$; $p < 0.01$). Except for day 12, no significant difference was observed in diet consumption between Green Muscle and Green Guard. A similar trend was observed in frass production, with significantly lower frass production in the treated groups compared to the controls ($F_{4,29} \le 104$; $p < 0.01$). Except for day 12, Green Muscle led to significantly lower frass production compared to the rest of the treatments. As days progressed, there was a decline in weight gain per day by treated locusts, while weight gain increased in the controls. Except at days 6 and 9, the effects of Green Muscle were not statistically significantly different from those of Green Guard (Figure 4a-c).

## 3.4. Greenhouse Trial

In the greenhouse trial, mortality in the controls was very low, and was <10% even at 15 days post-application, while mortality from all of the EPF was much higher (Figure 5). Green Muscle caused the highest mortality of third- and fifth-instar nymphs, but for adults, mortality was similar for Green Muscle and Green Guard. Mortality was less for the other two EPF, with mortality from *M. anisopliae* the lowest.
<figure><img src="image_124.png" /><figcaption>(b)</figcaption></figure>

<figure><img src="image_125.png" /><figcaption>(c)</figcaption></figure>

<figure><img src="image_126.png" /></figure>

**Figure 4.** Means (±SE) for (a) diet consumption, (b) frass production, and (c) observed weight of fifth-instar *S. gregaria* treated with a sublethal dose (1 × 10⁴ conidia/mL) of Green Muscle, Green Guard, *B. bassiana*, or *M. anisopliae*. For each subfigure: different lowercase letters indicate significant differences between different EPF; different uppercase letters indicate significant differences between different days (Tukey's HSD test at *p* ≤ 0.05).

As with the laboratory assay, the greenhouse bioassay demonstrated that 3rd-instar nymphs were most susceptible, followed by 5th-instar nymphs, and then adults (Figure 5a-c).
<figure><img src="image_127.png" /><figcaption>**Figure 5.** Mean mortality percentages (±SE) of (a) 3rd-instar, (b) 5th-instar, and (c) adult *S. gregaria* inoculated with $1 \times 10^9$ conidia/mL of Green Muscle, Green Guard, *B. bassiana*, or *M. anisopliae* in a greenhouse. Within each subfigure and between the treatments, different letters indicate significant differences (Tukey's HSD test at *p* ≤ 0.05).</figcaption></figure>

### 3.5. Field Trial

For EPF sprayed with a knapsack sprayer in field cages, all of the EPF produced significantly higher mortality ($F_{4,29} \le 49.6$; *p* < 0.01 in 3rd-instar nymphs; $F_{4,29} \le 69.9$; *p* < 0.01 in 5th-instar nymphs; and $F_{4,29} \le 133.0$; *p* < 0.01 in adults) compared to untreated controls. Green Muscle caused the greatest mortality among all developmental stages, with mortality from Green Guard statistically slightly lower. As with the other assays, the field bioassay demonstrated that 3rd-instar nymphs were the most susceptible (Figure 6a-c).
<figure><img src="image_128.png" /><figcaption>**Figure 6.** Mean mortality percentages (±SE) of (a) 3rd-instar, (b) 5th-instar and (c) adult *S. gregaria* sprayed with 1 × 10⁹ conidia/mL of Green Muscle, Green Guard, *B. bassiana*, or *M. anisopliae* using a knapsack sprayer in field cages. Within each subfigure and between the treatments, different letters indicate significant differences (Tukey's HSD test at *p* ≤ 0.05).</figcaption></figure>

**4. Discussion**

In the present study, laboratory, greenhouse, and field trial results showed that all of the formulated products (Green Muscle, Green Guard, and *M. anisopliae* (CQMCC No. 0877)) and the local isolate of *B. bassiana* caused significant mortality of *S. gregaria*. Green Muscle performed best, with the greatest mortality, followed by Green Guard, *B. bassiana*, and *M. anisopliae*. Third-instar nymphs were more susceptible than fifth-instar nymphs or adults. Experiments in greenhouse and field conditions showed that all of the tested EPF have the potential to reduce pest populations under natural conditions. Previous studies showed significant efficacy of Green Muscle, Green Guard, and *B. bassiana* against different species of locust and grasshopper, and the present study showed that all of the EPF caused significant mortality of various stages of *S. gregaria*. As in our studies, 3rd-instar nymphs were found to be more susceptible to EPF by both Bashir and El Shafie [30], who treated desert locusts of various stages with *M. acridum*, and Youssef et al. [31], who treated desert locusts with *B. bassiana* and *Entomophthora* sp. Additive and synergistic interaction was
observed when lower and higher doses of *M. acridum* and *Paranosema locustae* were tested against 5th-instar *S. gregaria* nymphs under laboratory conditions [28].

The current study is the first report on sublethal effects of EPF on the reproduction ability, fertility, and subsequent developmental stages of *S. gregaria*. Even though the applied dose was 1/10th of a dose causing 40% mortality, and 1/10,000th of the dose causing 80% mortality, infected females produced fewer egg pods and fewer eggs per pod, and fewer of the eggs that were produced actually hatched. Increased developmental duration was observed in offspring nymphs, while reduction in adult longevity was also detected. Effects on reproduction have been found in the past, but these were at a dose that was eventually lethal. Fewer egg pods per female and a lower total number of eggs per pod were observed in *S. gregaria* and in the lamenting grasshopper *Eyprepocnemis plorans* (Charpentier) when infected with *B. bassiana* and *M. acridum* [39], and in *Dichroplus maculipennis* (Blanchard) and *Ronderosia bergi* (Stål) infected with *B. bassiana* [40]. A 21–53% reduction in egg pods per female was observed for Moroccan locusts *Dociostaurus maroccanus* (Thunberg) (Orthoptera: Acrididae) infected with *M. acridum* (IMI 380189) and *B. bassiana* [53]. Fungal infection seems to utilize host resources to such an extent that there are less resources available for egg maturation [33,57]. Contrary to our findings was a report by Blanford and Thomas [27], who did not observe any sublethal effect of *M. acridum* (IMI 330189) on *S. gregaria* reproduction. Furthermore, we have for the first time detected the effect of EPF on the fertility or hatching of eggs of this species. Lower egg fertility was observed in *D. maroccanus* when infected with fungal isolates [53].

Sublethal EPF infections not only reduced reproductive capability, but also led to behavioral changes, including reduction in food consumption, reduced fecal production, and reduced weight. Upon infection with EPF, reduction in feeding was observed, similar to the observations of Moore et al. [35] of a reduction in diet consumption in both *S. gregaria* and the Central American locust *S. piceifrons* starting three days post-infection. Likewise, Mohammadbeigi and Port [34] observed a reduction in the diet consumption of *Uvarovistia zebra* (Uvarov) (Orthoptera: Tettigoniidae) infected with fungi. In the present study, infected individuals had lower fecal production compared to controls. A reduction in fecal production in *D. maroccanus* was observed from all of the fungal treatments except EABb 90/2-Dm [53]. Reduction in weight was observed among the different EPF compared to controls but, contrary to our findings, a previous study [30] reported no significant difference in the weight of controls and individuals treated with neem oil and *M. acridum*.

In this study, we observed significant efficacy of EPF not only in the laboratory, but also in the greenhouse and field experiments, despite several environmental factors that may restrict the efficacy of these pathogens under natural conditions [47]. A most important factor is ultraviolet (UV) radiation, which directly reduces pathogen survival, and also enables locusts to increase their body temperature and produce immunity against fungal infection [58]. The current study revealed that in spite of thermoregulation, all of the EPF have the ability to cause mortality, indicating their potential as biocontrol agents in the field. Our results are consistent with [15], where it was found that Green Muscle was effective in controlling locust adults, with some added advantages compared to chemicals. As with the greenhouse experiment, in field conditions these results show that EPF have great efficacy against *S. gregaria*. Future research will focus on investigating the compatibility of EPF with other control agents, including entomopathogenic nematodes, chemical insecticides, and botanical extracts, as these have been found to be effective against a variety of insect pests [30,49,50,59–61].

**5. Conclusions**

The present research shows that Green Muscle, Green Guard, *B. bassiana*, and *M. anisopliae* have the potential to control locusts under field conditions. In addition, there are sublethal effects on their reproductive ability and behavior, with reduction in diet consumption, frass production, and weight observed. Future research needs to explore the combination of EPF with other control agents—including chemical insecticides, mi-
crosporidia, and botanical extracts—as a means of integrated pest management of locusts in the field.

**Author Contributions:** Conceptualization, W.W.; research design, W.W.; methodology, W.W., M.U.G., D.H. and W.S.; data curation, W.W.; formal analysis, W.W. and M.U.; performed research, W.W. and M.U.G.; visualization, W.W.; writing—review and editing, W.W., M.U.G., D.H. and W.S.; supervision, W.W.; funding acquisition, W.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the National Disaster Management Authority (NDMA), Government of Pakistan, Islamabad, Pakistan.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are contained within the article.

**Acknowledgments:** The authors are thankful to Atif Kamal and Murtaza Quddusi (BASF Pakistan Pvt. Ltd.) for providing Green Guard, Yann Miège (Elephant Vert, Morocco) for Green Muscle, and Zhongkang Wang (Chongqing Chongda Bio-Tech Development Ltd. Co., China) for *M. anisopliae* (CQMCC No. 0877). We are thankful to Tariq Butt (Swansea University, Swansea, UK) and Syed Farooq Shah (Razbio Ltd., UK) for their technical support in this study. This study was financially supported by the National Disaster Management Authority (NDMA), Government of Pakistan, Islamabad, Pakistan.

**Conflicts of Interest:** The authors declare no conflict of interest.

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Review

# Nosema locustae (Protozoa, Microsporidia), a Biological Agent for Locust and Grasshopper Control

Long Zhang<sup>1,2,*</sup> and Michel Lecoq<sup>3 ID</sup>

**Abstract:** Effective locust and grasshopper control is crucial as locust invasions have seriously threatened crops and food security since ancient times. However, the preponderance of chemical insecticides, effective and widely used today, is increasingly criticized as a result of their adverse effects on human health and the environment. Alternative biological control methods are being actively sought to replace chemical pesticides. *Nosema locustae* (Synonyms: *Paranosema locustae*, *Antonospora locustae*), a protozoan pathogen of locusts and grasshoppers, was developed as a biological control agent as early as the 1980s. Subsequently, numerous studies have focused on its pathogenicity, host spectrum, mass production, epizootiology, applications, genomics, and molecular biology. Aspects of recent advances in *N. locustae* show that this entomopathogen plays a special role in locust and grasshopper management because it is safer, has a broad host spectrum of 144 orthopteran species, vertical transmission to offspring through eggs, long persistence in locust and grasshopper populations for more than 10 years, and is well adapted to various types of ecosystems in tropical and temperate regions. However, some limitations still need to be overcome for more efficient locust and grasshopper management in the future.

**Keywords:** locust; grasshopper; biological control; *Nosema locustae*; application; epizootics

## 1. Introduction

Locust and grasshopper (L&G) outbreaks, often resulting in huge plagues, have been a serious threat to global food security since ancient times [1]. Traditional control of L&G consists mainly of the application of chemical pesticides, which often results in many side effects. These include toxic chemical residues on food, adverse health effects on humans and nontarget animals, and environmental pollution. One of the most promising alternatives to chemical pesticides is biological control. Although there are many natural enemies of L&G [2–4], only a few have been developed as biological control agents or potential agents, including the microsporidian *Nosema locustae* (synonym: *Antonospora locustae*, *Paranosema locustae*) and the fungus *Metarhizium acridum*, both of which have been quite widely used in the control of L&G.

*N. locustae*, a unicellular eukaryote (Figure 1) with an obligate intracellular lifestyle [5–7], was the first to be commercially developed for L&G control since its discovery and characterization of its potential as a biological control agent against these species [8–10]. Due to its slow action and various other constraints, *N. locustae* was considered to be of limited application [11,12]. However, as the only microsporidian agent for L&G control, *N. locustae* has been studied extensively [1,13–17]. In recent years, there has been a renewed interest in *N. locustae*, mainly due to work in China, where it is produced in large quantities and used extensively, and Argentina, where its long-term persistence appears to reduce the frequency and intensity of grasshopper outbreaks [18]. Recent advances in areas such as mass production, formulation, application, and epizootiology have

**Citation:** Zhang, L.; Lecoq, M. *Nosema locustae* (Protozoa, Microsporidia), a Biological Agent for Locust and Grasshopper Control.
*Agronomy* **2021**, *11*, 711. https://doi.org/10.3390/agronomy11040711

Academic Editor: Andrea Sciarretta

Received: 23 February 2021

Accepted: 2 April 2021

Published: 8 April 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

<figure><img src="image_129.png" /></figure>

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

<footnote n="1">¹ Shandong Academy of Agricultural Sciences, Jinan 250100, China</footnote>

<footnote n="2">² Department of Grassland Resources and Ecology, College of Grassland Science and Technology, China Agricultural University, Beijing 100193, China</footnote>

<footnote n="3">³ CIRAD, UMR CBGP, F-34398 Montpellier, France; mlecoq34@gmail.com</footnote>

<footnote>* Correspondence: locust@cau.edu.cn</footnote>
further promoted the application of this pathogen. Numerous studies on its host spectra,
molecular biology, and evolution have provided a solid fundamental basis for the use of
*N. locustae* for L&G management.

<figure><img src="image_130.png" /><figcaption>Figure 1. Spores of *N. locustae* under scanning electron microscopy (photo by Long Zhang).</figcaption></figure>

## 2. Host Spectrum

*N. locustae* was first identified in a laboratory population from the migratory locust *Locusta migratoria* (Linnaeus, 1758) by Elizabeth Canning in 1953 [19]. This pathogen occurs under natural conditions and has been found in various areas of the United States (Montana, Northern Dakota, Minnesota, Oregon, Wyoming, Colorado, Arizona, and Idaho), Canada (Saskatchewan, Ontario) and South America (western Pampas and northwestern Patagonia, Argentina), as well as Asia (Rajasthan and Vidarbha, India; Inner Mongolia, Hainan and Qinghai, China) and Africa (Karoo, South Africa) [20]. The highest level of infection was reported of about 5.5% in *Melanoplus sanguinipes* (Fabricius, 1798) in Idaho between 1963 and 1967 [21].

*N. locustae* has a wide host range restricted to orthopteran insects. Henry [22] in 1969 provided an initial list of 55 North American species that he knew to be susceptible. Brooks [15] published in 1988 a new list of worldwide susceptible orthopterans, expanding the host range to 95 species. The last review was done in 2005 by Lange [20], who arrived at a total of 121 species. Since then, various authors have been added to this list (Table 1), and we now reach a total of 144 susceptible orthopterans.
<table><caption>**Table 1.** List of orthopteran species infected by *N. locustae* but not included in Lange [20].</caption><thead><tr><th>Species</th><th>Inoculation Infection (Caging)</th><th>Field Trial Infection</th><th>References</th></tr></thead><tbody><tr><td colspan="4"><strong>Asia</strong></td></tr><tr><td>Bryodemella holdereri (Krauss, 1901)</td><td>X</td><td></td><td>[23]</td></tr><tr><td>Calliptamus italicus (Linnaeus, 1758)</td><td>X</td><td></td><td>[24]</td></tr><tr><td>C. abbreviatus Ikonnikov, 1913</td><td>X</td><td></td><td>[23]</td></tr><tr><td>Ceracris kiangsu Tsai, 1929</td><td>X</td><td>X</td><td>[25]</td></tr><tr><td>Chondracris rosea (De Geer, 1773)</td><td>X</td><td>X</td><td>[26]</td></tr><tr><td>Chorthippus dubius (Zubovski, 1898)</td><td></td><td>X</td><td>[27]</td></tr><tr><td>C. brunneus (Thunberg, 1815)</td><td>X</td><td></td><td></td></tr><tr><td>Damalacantha vacca (Fischer von Waldheim, 1846)</td><td>X</td><td></td><td>[23]</td></tr><tr><td>Deracanthella aranea (Fischer von Waldheim, 1833)</td><td>X</td><td></td><td></td></tr><tr><td>Dociostaurus kraussi (Ingenitskii, 1897)</td><td>X</td><td></td><td>[24]</td></tr><tr><td>Fruhstorferiola tonkinensis (Willemse, 1921)</td><td></td><td>X</td><td>[28]</td></tr><tr><td>Gampsocleis sedakovii (Fischer von Waldheim, 1846)</td><td>X</td><td></td><td>[23]</td></tr><tr><td>Haplotropis brunneriana Saussure, 1888</td><td>X</td><td></td><td></td></tr><tr><td>Oedaleus decorus (Germar, 1825)</td><td>X</td><td></td><td>[24]</td></tr><tr><td>Arcyptera meridionalis Ikonnikov, 1911</td><td>X</td><td>X</td><td></td></tr><tr><td>Sphingonotus mongolicus Saussure, 1888</td><td>X</td><td></td><td>[23]</td></tr><tr><td colspan="4"><strong>Africa</strong></td></tr><tr><td>Acrotylus blondeli Saussure, 1884</td><td></td><td>X</td><td></td></tr><tr><td>Acrotylus patruelis (Herrich-Schäffer, 1838)</td><td></td><td>X</td><td></td></tr><tr><td>Aiolopus thalassinus (Fabricius, 1781)</td><td></td><td>X</td><td>[29]</td></tr><tr><td>Anacridium melanorhodon (Walker, 1870)</td><td></td><td>X</td><td></td></tr><tr><td colspan="4"><strong>America</strong></td></tr><tr><td>Amblytropidia australis Bruner, 1904</td><td></td><td>X</td><td></td></tr><tr><td>Dichroplus vittigerus (Blanchard, 1851)</td><td></td><td>X</td><td>[30]</td></tr></tbody></table>

Although *N. locustae* has a broad host spectrum in Orthoptera, it is incapable of affecting non-orthopteran insects. It has been shown that the honey bee (*Apis mellifera*) and the lepidoterans *Heliothis zea* and *Agrotis ipsilon* were not susceptible [15]. Menapace et al. [31] reported that honey bees were not infected, even when fed high doses of spores. The American cockroach *Periplaneta americana* and the spider *Butalus occidentalis* were also found to be non-susceptible [32]. *N. locustae* has been demonstrated to be safe for vertebrates. Brooks [15] summarized the assessment tests on primary skin irritation, acute dermal toxicity, acute inhalation toxicity and pathogenicity, subacute oral toxicity, acute oral toxicity, acute pathogenicity, and possible hazards of *N. locustae* to vertebrates, including rabbits, guinea pigs, rainbow trout, giant toads, mallards, ring-necked pheasant, mice, and rats. No significant effects were observed. The toxicity of *N. locustae* at 20 million spores/mL to certain non-target organisms, such as *Coturnix japonica*, *Apis mellifera*, *Bombyx mori*, *Daphnia magna*, and *Brachydanio rerio*, was examined in China. The results showed that there was no risk to honey bees per os at the maximum exposure dose tested, no deaths in the five animal species tested in the contact toxicity experiments, and *N. locustae* was relatively safe for nontarget beneficial organisms in the environment [33].

### 3. Pathogenicity

*N. locustae*, as an obligate parasite, reproduces in host target cells. Its infection involves the polar tube of the spore to inject its plasma into the target cells [5]. It causes high mortality in L&G. The locust's main target organ is the host's adipose tissue (fat body) [9]. *N. locustae* penetrates fat body cells and produces meronts, sporonts, sporoblasts, and spores. In the migratory locust, *Locusta migratoria migratoriaoides* (Reiche & Fairmaire, 1849), the younger the nymphs, the more susceptible they are, but even newly emerged adults are still susceptible [9]. Studies by Tounou et al. [34] on the effects of *N. locustae* on the desert locust, *Schistocerca gregaria* (Forskål, 1775) and the Senegalese grasshopper, *Oedaleus senegalensis* (Krauss, 1877), showed that *N. locustae* has high pathogenicity on the young nymphal instars of these two species. The median survival time for first, second, third,
fourth, and fifth nymphal instars was 6, 9, 10, 14, and 15 days, respectively, when locusts were inoculated with $1 \times 10^7$ spores on 10 g wheat bran in groups. Similar results were obtained with the Senegalese grasshopper using the same treatment method, with median survival times for instars 1, 3, and 5 being 5, 9, and 15 days, respectively. The median survival time therefore increased with the age of the locust and with decreasing inoculation doses. For instance, third-instar desert locust nymphs were inoculated in groups with $5.62 \times 10^6$ or $3.16 \times 10^4$ spores with a median survival time of 14 and 16 days, respectively. However, cumulative mortality increases with the increase of spore concentration and decreases with the increasing age of the nymphs. *N. locustae* caused high mortality in young desert locust nymphs. Mortality was 100% in first and second nymphal instars inoculated in groups with $1 \times 10^7$ spores of *N. locustae*, as well as in first nymphal instars with $1 \times 10^6$ spores. With third and fourth nymphal instars inoculated with $1 \times 10^7$ spores, cumulative mortality was above 90%. Similar results were obtained with the Senegalese grasshopper, which was inoculated with $1 \times 10^7$ spores: 100% mortality was observed in the first instar but 88.5% mortality in the third instar. In the fifth instar, mortality was only 66.3% in desert locust and 70% in the Senegalese grasshopper. These results suggest that the appropriate period for the application of *N. locustae* is mainly between the first and fourth instars, i.e., a possible application period of about 20–28 days, taking into account an average duration of 5–7 days per nymphal instar.

Inoculation of the 1st–5th nymphal instars of *Chondracris rosea* (De Geer, 1773) with *N. locustae* resulted in 78.2% to 100% mortality in field caging experiments by Liu and Chen [26]. The LC₅₀ was approximately $3.88 \times 10^5$ spores/mL for the 1st–3rd instars and $3.98 \times 10^6$ spores/mL for the 4th–5th instars. In a caging experiment on a large forest site, the same authors observed that *C. rosea* mortality was greater than 91.1% when they sprayed *N. locustae* at a rate of $5 \times 10^7$ spores/mL. At $1 \times 10^8$ spores/mL, mortality reached 100% 25 days after treatment [26]. Chen et al. [25] conducted a laboratory experiment with five concentrations of *N. locustae* spores ($1 \times 10^4$, $1 \times 10^5$, $1 \times 10^6$, $1 \times 10^7$, and $1 \times 10^8$ spores/mL) to treat early stages (1st–2nd instar) of the yellow-spined bamboo locust (*Ceracris kiangsu*). Mortalities were 16.6, 32.9, 29.2, 34.0, and 83.7%, respectively, increasing with spore concentrations. They also found that yellow-spined bamboo locust mortality was 85.1% after the application of a suspension of *N. locustae* spores at a concentration of $5 \times 10^7$ spores/mL in field trials in the forest. Zang et al. [35] reported that mortality of *Oxya chinensis* (Thunberg, 1815) was about 65.4–68.1% 30 days after the field treatment of third-instar nymphs sprayed with $1.5 \times 10^{10}$, $2.25 \times 10^{10}$ spores/ha, respectively. The infection rate in survivors was approximately 40%.

Concurrent use of *N. locustae* and *Metarhizium* spp. has shown additive effects on locusts and grasshoppers. When fifth-instar nymphs of *S. gregaria* were inoculated first with *N. locustae* at doses between $1 \times 10^4$ and $1 \times 10^6$ spores on wheat bran in groups, and then 10 days later with *M. anisopliae* at doses between $1 \times 10^2$ and $1 \times 10^4$ spores/nymph, the median survival times ranged from 3 to 9 days, and the shortest duration was only 3 days at doses of $1 \times 10^6$ spores of *Nosema* and $1 \times 10^4$ spores of *Metarhizium* [29]. When the locusts were inoculated in groups with *N. locustae* at doses of $1 \times 10^5$ and $1 \times 10^6$ spores, and *Metarhizium* at $1 \times 10^3$ and $1 \times 10^4$/nymph, the mortality was at 90% and 97% over 3 days, and both reached 100% 10 days after inoculation. Mortality was only 12.2% in the control, showing a synergistic effect between these two agents. Similar results were obtained with *N. locustae* directly mixed with *Metarhizium* spp., with the oriental migratory locust (*L. migratoria manilensis* Meyen, 1835). Mortality was higher with the mixture compared to treatments with each agent applied separately. The effects of a mixture of *M. acridum* and *N. locustae* on third nymphal instars under laboratory conditions showed that at a ratio of 1:1 (*M. acridum* at $3.90 \times 10^6$ spores/g locust body weight and *N. locustae* at $3.99 \times 10^6$ spores/g locust body weight), locust mortality was about 96.7% 24 days after inoculation, showing an additive effect of these two agents [36]. When 2nd–3rd-instar nymphs of *L. migratoria* were inoculated with $1 \times 10^6$ spores/nymph of *N. locustae*, and after 3, 6 and 9 days with *Metarhizium* at $1 \times 10^7$ conidia/mL, an additive effect was only
observed in nymphs inoculated with *Nosema* and then 9 days later with *Metarhizium* [37]. After examining the stage of *Nosema* development on Days 3, 6, and 9 after inoculation, they found that it was not until the *Nosema* spore maturation stage that locusts were most susceptible to fungal infection.

Lv et al. [38] identified 4 defensins from migratory locust palp transcriptomes, named LmigDEF1 (78 amino acids), LmigDEF3 (78 amino acids), LmigDEF4 (69 amino acids), and LmigDEF5 (67 amino acids) with theoretical isoelectric points (pI)/molecular weights (Mw, kDa) of 6.48/8.29, 6.56/8.37, 8.23/7.19, and 8.27/6.93, respectively. The expression patterns of LmigDEF1, LmigDEF3, and LmigDEF5 in the fat body and salivary glands were examined by qRT-PCR after the locusts were inoculated with *N. locustae*. Results indicated that all three defensins varied over time in the fat body and salivary glands after *Nosema* infection, the transcript level of the LmDEFs being at their lowest in the fat body over the 10 days. This indicates that *Nosema* infection reduced the locust's immune response via the defensins and may explain why the coordinated use of *Nosema* and *Metarhizium* results in higher locust mortality [29,36]. In particular, for about four days when *Nosema* were sporulating after inoculation, the locusts were more easily infected by the fungus due to the lower level of the three types of defensins in the fat body [37]. In addition, Chen et al. [39] demonstrated the key role of *N. locustae* sporulation in locust mortality. They identified a spore wall protein, AlocSWP2 from *N. locustae*, containing four cysteines. AlocSWP2 has been detected in the wall of mature spores, sporoblasts, and sporonts during sporulation in the host body by immunocytochemistry localization experiments. AlocSWP2 was detected in the fat body of infected locust only on Day 9 after inoculation using RT-PCR. The survival percentage of infected locusts that received a dsRNA injection of AlocSWP2 on Days 15, 16, and 17 after inoculation of *Nosema* spores was significantly higher than that of infected locusts without dsRNA treatment. Similarly, the number of spores in locusts infected with *Nosema* and treated with RNAi of AlocSWP2 was significantly lower than that in infected locusts without RNAi of this gene. This indicates that this *N. locustae* spore wall protein is involved in sporulation, contributing to host mortality.

*L. migratoria migratorioides* infected with *N. locustae* demonstrated reduced sustainable flight capacity [9]. Zhang et al. [40] confirmed this with the oriental migratory locust (*L. migratoria manilensis*). Flight capacity of infected and healthy adult locusts, 5 to 15 days after emergence, was determined using a flying mill for 18 h. On average, the flight distance of healthy versus infected locusts was 14,279 vs. 864 m; flight speed, 1.23 vs. 0.51 m/s, flight time, 3 vs. 0.33 h; maximum flight distance, 72,538 vs. 1544 m; and maximum sustained flight time, 6.7 h vs. 0.1 h. The decrease in flight capacity may be due to the fact that *N. locustae* destroyed the fat bodies reducing the supplement of glyceride and fat as energy resources. In *L. migratoria manilensis* infested with *N. locustae*, the glyceride content decreased rapidly, while lipase activity increased in both hemolymph and total fat [41].

Locusts and grasshoppers infected with *N. locustae* have reduced fertility [13,42]. Reduced vitellogenin was observed in fourth-instar nymphs of *L. migratoria manilensis* inoculated with *N. locustae*: vitellogenin levels in the fat body, hemolymph, and ovaries were very low compared to the control [43]. The maximum vitellogenin level in infected vs. healthy locusts, respectively, were 4.663 vs. 18.655 mg/mL in the fat body, 2.627 vs. 7.603 mg/mL in the hemolymph, and 4.927 vs. 73.367 mg/mL in the ovaries. This explains why the reproductive capacity of infected locusts is low.

The disease caused by *N. locustae* is transmitted vertically in eggs and egg pods [5]. Infected females have been reported to lay eggs containing spores of *N. locustae* [42]. Raina et al. [44] reported vegetative stages of *N. locustae* in the yolk of the oocyte and spores in the eggs of *L. migratoria*. When fourth-instar nymphs were inoculated with a dose of 1.5 × 10⁶ spores, the infected parents laid eggs and the prevalence of infection was 100% in the next generation. The disease was vertically transmitted up to 14 generations, and mortality due to vertical transmission at times reached more than 90%. Parents of *S. gregaria* and *O. senegalensis* infected with *N. locustae* produced progeny with a 50% infection rate, indicating high vertical transmission in these species [34].