Prevalence rates of microsporidia in locusts and grasshoppers in South-Western Russia

Locusts and grasshoppers are dangerous polyphagous pests of agricultural crops. In the present paper, results of screening of Acridoidea populations in the South-Western Russia for microsporidia infections including locusts Locusta migratoria, Dociostaurus maroccanus, and Calliptamus italicus and grasshoppers Chorthippus loratus, Oedipoda caerulescens, and Acrida bicolor, are presented. Microsporidia prevalence rates were estimated using light microscopy of fresh smears. Out of 179 specimens of L. migratoria sampled between 2002 and 2019 in Krasnodar Territory, Astrakhan and Rostov Regions, none was infected with microsporidia. Similarly, 95 specimens of D. marrocanus from Krasnodar Territory (2017) and Dagestan Republic (2009) were also negative for microsporidia. Meanwhile, one positive case was detected for C. italicus corresponding to 0.5 % for the total amount of 192 exemplars collected from 2002 to 2019 in Krasnodar Territory, Astrakhan and Rostov Regions. As for grasshoppers, all Ch. loratus samplings in Krasnodar Territory in 2017-2019 were infected at the prevalence rates of 2.2-15 %, though no infection was found in 40 specimens in Crimea in 2019. In 56 individuals of O. caerulescens collected from Rostov Region and Krasnodar Territory, the microsporidia prevalence rate was 1.8 %. Among 96 specimens of A. bicolor, none was infected. In total, the microsporidia prevalence rates were higher in grasshoppers as compared to locusts, the difference being statistically significant at p<0.01.


Introduction
Modern plant protection implies the management of populations of organisms harmful to plants in agricultural ecosystems. Phytosanitary monitoring is among the most important prerequisites for effective pest management. Phytosanitary forecasts must meet the requirements of reliability, quality and accuracy, taking into consideration the effects of regulatory mechanisms as the key prognostic criterion, rather than merely a correction factor [1].
Locusts and grasshoppers (Acridoidea) are the most dangerous polyphagous agricultural pests. Huge territories of floodplain zones for centuries serve as a reservation of migratory locust, Italian locust and grasshoppers dwelling in the natural environment on perennial grasses, roadsides and uncultivated lands. Over the past 20 years, outbreaks of locusts have been reported in the South of Russian Federation and the Republic of Kazakhstan, leading to enormous crop losses of a wide range of cultivated plants [2].
However, the representative of the genus Paranosema should be recognized as the most promising for the development of microbial formulations to control a number of pests. Discovered in a culture of migratory locusts Locusta migratoria, the microsporidium Paranosema locustae (Antonospora, Nosema) [9] was found able to infect over 120 host species among Orthoptera [10,11] and to retain infectivity for years [12]. In 1980, formulations were designed and registered in US (Nolo Bait™, Semaspore™) against Orthoptera, remaining the only mean for pest control based on microsporidia [13].
Traditionally, the use of entomopathogens, including those for locust control, is carried out similarly to the application of chemical insecticides, i.e. by treating pests in the active phases of development, without taking into account further circulation of the pathogen in the host insect population [14]. For a long-term decline in numbers, it seems relevant to create long-term infectious foci in the biotopes inhabited by harmful insects, primarily in their reservation sites. This approach can be effective for obligate parasites, well adapted to the permanent presence in host populations. For example, long-term (over 10 years) circulation after introduction in local populations of grasshoppers has been shown for P. locustae in Argentina [15].
Screening of locust and grasshoppers populations for parasitic microorganisms is important for understanding factors and patterns of density dynamics, to reveal novel isolates which may serve as promising producers of microbial formulations and to track previously released pathogens. The goal of the present study is access and compare prevalence rates of microsporidia in locusts and grasshoppers in their natural habitats in South-Western Russia.

Materials and Methods
To reveal microsporidia infections causing diseases of Acrididae, screening of populations of locusts and grasshoppers was performed in the South-Western Russia. Insects were caught between 2002 and 2019 by net or by hand in ten sampling sites (Fig. 1).
Locusts of the species Locusta migratoria, Dociostaurus maroccanus, and Calliptamus italicus and grasshoppers of the species Chorthippus loratus, Oedipoda caerulescens, and Acrida bicolor were sampled and maintained under laboratory conditions in cages, fed with foliage of monocotyledonous crops and weeds, including Zea mais, Triticum durum, Poa pratensis etc. Insects perished during transportation and maintenance were stored as dried cadavers at ambient temperature.  (Table 1). To summarize, overall prevalence rate of microsporidia in the three locust species reached 0.2 % (N=466, Table 2).
Notably, prevalence rate differences between total locust and grasshopper samplings were statistically significant at p<0.01 (Table 2). It can be concluded that populations of grasshoppers are infected with mcirosporidia at higher degree as compared to the locust populations. This could be influenced by various environmental factors associated with the properties of both parasites and insect hosts, as well as environmental conditions. In particular, it is known that fever can directly kill the infection in the body of insects and also stimulate the immune system and increase resistance to disease; different types of insects have a certain innate level of resistance to various pathogens; biological properties of the pathogen affect its adaptability to a particular insect species, including virulence, ability to horizontal and vertical transmission, etc. [23,24].
Among the examined species of Acridoidea, the highest level of infection with microsporidia was observed in Ch. loratus. This is also noteworthy that insects of this species captured in the summer period were infected to a lesser extent as compared than insects captured in the autumn period. The microsporidia prevalence rate in autumn samples was 2.5 times higher than in summer samples.