Characterization, distribution, and virulence of protistan entomopathogen, Mattesia dispora (Sporozoa, Gregarina) in the Indian meal moth, Plodia interpunctella (Lepidoptera: Pyralidae) populations in Turkey

Indian meal moth, Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae) is the dominant pest on the stored products throughout the world. As an alternative to chemical insecticides, entomopathogens can be natural suppressors for pest populations. For this reason, the study of entomopathogens existing in the natural population of a pest contributes to the decision-making process of controlling that pest. In the present study, characterization, distribution, and virulence of protistan entomopathogen, the Turkish strain of Mattesia dispora in the Indian meal moth, Plodia interpunctella populations were presented. During the microscopic observations, a protistan entomopathogen was found in the populations of P. interpunctella in Turkey. It was identified as the Turkish strain of Mattesia dispora, a neogregarine pathogen. Typical fresh navicular oocysts of the pathogen were 13.28 ± 0.41 (13.1–14.41) μm in length and 7.72 ± 0.51 (6.6–8.54) μm in width (n = 50). Oocysts stained with Giemsa measured 12.32 ± 0.78 (10.88–13.24) μm in length and 7.01 ± 0.26 (6.5–7.43) μm in width. Polar plugs were recognizable clearly by light and electron microscopy, measuring 900 to 1100 nm. The oocyst wall was quite thick, measuring 600 to 800 nm. Each oocyst contained 8 sporozoites. 2.047 dead and 413 living larvae, 932 adults, and 40 pupae, collected from 14 different locations from 2019 to 2021 were examined for the presence of the protistan entomopathogen. In total, 225 of 3.432 P. interpunctella adult and larvae were found to be infected with this pathogen. Total infection occurred as 5.2 for M. dispora. Infection rates by M. dispora were 4.8% for dead larvae, 14.8% for living larvae, and 2.1% for adults. On the other hand, M. dispora infections reached 33% in some populations. M. dispora infections were observed in the seven (50%) of the examined populations. Furthermore, the Turkish strain of M. dispora had a high pathogenic effect against the second/third instar larvae of P. interpunctella. The average mortality rate was 98.33%. Little is known about neogregarine infections as a natural suppressing factor in pest populations. The Turkish strain of M. dispora is very common and widespread in the populations of P. interpunctella. Furthermore, it has very high virulence on the P. interpunctella larvae. Such a widespread infection and very high virulence are desirable properties for a biological control agent. The results indicate that M. dispora can be an important natural suppressing protistan entomopathogen in P. interpunctella populations.


Background
The Indian meal moth, Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae), is the dominant pest on the stored products throughout the world. Synthetic insecticides applied to stored products or fumigation are used to reduce losses of stored products (Freitas et al. 2020). However, it has been realized that these insecticides are not quite innocent over the years. Because they pose a great risk to both nature and beneficial insects as well as humans. Therefore, there is an urgent need to develop an alternative safe pest control strategy with less harmful effects on humans and the environment (Kumar et al. 2012), which is possible with entomopathogenic organisms (Baki et al. 2021;Yaman et al. 2021). Entomopathogens that cause disease in pests are natural suppressors of pest populations. For this reason, the study of entomopathogens existing in the natural population of a pest contributes to talking the decision-making process to control that pest.
There is a new interest in using entomopathogens for microbial control of P. interpunctella as well as other stored product pests . Detection of different natural pathogens and parasites of P. interpunctella for controlling the population can be most successful in the biological control of this pest. Among the entomopathogens, protistan entomopathogens are often prevalent and persistent in natural populations of pest insects, studies on their use as potential microbial insecticides have generally been limited due to their high host specificity and difficulties with mass production. In addition, their suppressive potential in natural populations has not been adequately studied. Some entomopathogenic protists such as microsporidia, neogregarines, and coccidia are known to infect storage pest insects, however, entomopathogens naturally occurring in P. interpunctella populations have not been enough investigated. In this study, characterization, distribution, and virulence of protistan entomopathogen, M. dispora in P. interpunctella were studied to document the natural suppressing potential of this pathogen in P. interpunctella populations.

Macroscopic-microscopic examination and characterization of protist pathogens
Macroscopic examination of a field-collected insect sample may offer some clues as to disease-causing entomopathogens in its natural populations. Therefore, the samples were firstly taken for macroscopic examination. After macroscopic examination, living and dead samples suspected with a symptoms of disease were separated individually for microscopic examinations. All samples were dissected in Ringer's solution and then prepared wet smears including host fat body, malpighian tubules, gut epithelium, and hemolymph were examined for the presence of protistan entomopathogens under a light microscope at a magnification of 400-1000×. When infection was found, the slides were air-dried and fixed with methanol, then stained with a freshly prepared 5% solution of Giemsa stain. They were then washed in running tap water, air-dried, and examined under a microscope (Yaman 2020). The oocysts of the protist pathogens detected by the light microscopy were measured and photographed using a microscope with a digital camera and soft imaging system. A part of the infected specimens was used for preparing samples for transmission electron microscopy (TEM) using previously reported techniques.

Bioassay tests for the potential of the isolated protistan entomopathogen
Virulence of the isolated protistan entomopathogen was tested against the second/third instar larvae of P. interpunctella. Oocyst of the pathogen was harvested from the infected larvae at the 10-14×10 6 oocysts/ml concentrations and diluted to 1.6 × 10 6 to obtain the required concentration for experimental treatments. Second/third instar larvae of P. interpunctella larvae were fed on nut tablets dipped into the neogregarine pathogen suspensions. Three replications (Experimental group 1, 2, and 3) of the experimental group and two replications (Control group 1 and 2) of the control were used. Each bioassay group was performed by 20 insect larvae under the same laboratory conditions. All tested groups were properties for a biological control agent. The results indicate that M. dispora can be an important natural suppressing protistan entomopathogen in P. interpunctella populations.
Keywords: Plodia interpunctella, Stored product pest, Mattesia dispora, Virulence, Distribution, Natural suppressor Page 3 of 6 Yaman et al. Egyptian Journal of Biological Pest Control (2022) 32:82 kept at 24-28 °C and 35-45% RH and 18:6 photoperiod of laboratory conditions for 21 days. Observations were recorded daily and dead larvae were removed immediately. Experimental bioassays were repeated 3 times on different days and data was corrected, using Abbott's formula (Abbott 1925).

Statistical analysis
A chi-square test was used to compare observed results. A p-value less than 0.05 was considered significant.

Results
After macroscopic observations, disease-suspected specimens from the infected colonies were ailing obviously with symptoms such as slow movement, loss of appetite and color change, and in certain numbers dying although optimum living conditions are provided. During the microscopic observations, a protistan entomopathogen was found in the populations of P. interpunctella in Turkey (Figs. 1 and 2). It was a neogregarine pathogen. The neogregarine pathogen was observed in only larvae and adults of P. interpunctella, not in the pupae. The infected tissue was the fat body and hemolymph of the host. All life cycle stages such as oocysts, micronuclear and macronuclear merozoites, and gamonts of the neogregarine pathogen were observed in the wet or stained smear preparations.
During the study, 3432 samples of P. interpunctella samples including larvae, adults, and pupae were dissected and searched for neogregarine infection in the 14 localities of Turkey from the years 2019-2021. 2047 dead and 413 living larvae, 932 adults, and 40 pupae were examined for the presence of the neogregarine pathogen, 180 of 3432 P. interpunctella adults and larvae were found to be infected by this pathogen. Total infection occurred at 5.24% (Tables 1 and 2).
Neogregarine infection was observed in 7 of the examined 14 populations. The average of neogregarine infections for all populations was found as 4.8% in dead larvae, 14.8% in living larvae, and 2.1% in adults (Table 2). However, neogregarine infections had reached levels that can be considered high in some populations, as significant as 33%.
On the other hand, the virulence of the protistan pathogen against P. interpunctella larvae was also determined. Bioassay tests showed that the protistan pathogen had a high pathogenic effect against the second/third instar larvae of P. interpunctella (Fig. 5). Two of the three experimental groups had a 100% mortality rate, while a 95% mortality rate was achieved in one experimental group. In contrast, one larval death was observed in only one of the control groups. The average mortality rate was determined as 98.33%.

Discussion
In this study, 14 sampling populations were included to represent the whole of Turkey, a neogregarine pathogen of P. interpunctella was detected for the first time. Morphological and ultrastructural results showed that the described neogregarine had the typical characteristics of members of the genus Mattesia (Family Lipotrophidae: order Neogregarinorida (Apicomplexa)). It closely resembles Mattesia dispora, first described from the larvae of the flour moth, Ephestia kuehniella by Naville (1930) Neogregarines occur naturally in lepidopteran pests. Some are highly pathogenic. So, have been recognized as potential biocontrol agents against lepidopteran pests. However, the use of protistan pathogenic species as a control agent should be in the early stages of development. At the same time, extensive research is required to be used as a protective agent (Dales 1994). Several studies on pathogens and parasites of stored-product pests, mainly have focused on the isolation and characterization of pathogenic microorganisms. A few of them were carried out on the protist pathogens of P. interpunctella. Until now, microsporidian pathogens, Nosema plodiae (Kellen and Lindegren 1973), Vairimorpha plodiae , neogregarine pathogen, Mattesia dispora (Wendell and Dicke 1964), gregarine pathogen, Leidyana sp. (Suzaki et al. 2006) were studied as a microbial pathogen in P. interpunctella. There is only one study on the distribution, occurrence, and potential of a microsporidium, Vairiomorpha plodiae in P. interpunctella under natural conditions ). However,   Page 5 of 6 Yaman et al. Egyptian Journal of Biological Pest Control (2022) 32:82 there are no other studies on the distribution and potential of protistan entomopathogens in the natural populations of P. interpunctella. In this study, the presence, distribution, and virulence of M. dispora in 14 populations of P. interpunctella were investigated. M. dispora was detected in 7 (50%) of the 14 populations examined. The fact that M. dispora infects half of the populations studied and its infection rate reaches up to 33.8% in some populations (Table 1) indicates that M. dispora can be an important natural suppressing protistan entomopathogen in P. interpunctella populations in Turkey. In addition, P. interpunctella larvae and adults were found to be infected by the neogregarine pathogen. As shown in Table 1, M. dispora is very common in the populations of P. interpunctella. Such a widespread infection is a desirable property for a biological control agent (Pereira et al. 2002). Additionally, among gregarines only the neogregarines had a high pathogenic effect on their hosts by destroying the host's fat body and exhausting energy sources. The results confirmed that M. dispora infections are desirable and significant natural suppressor factors in P. interpunctella populations.
In neogregarines, the members of the genus Mattesia are known as important pathogens of various insects with a significant pathogenic effect on their host (Valigurova and Koudela 2006). Therefore, their effects on several host insects have been investigated by several authors for microbial control. In an extensive study, the susceptibility of several insect pests of stored grain to 2 Mattesia species, M. oryzaephili isolated from Cryptolestes ferrugineus and M. dispora obtained from E. kuehniella were determined for microbial control (Lord 2003).
On the other hand, bioassay experiments showed that the Turkish strain of the neogregarine, M. dispora had a high virulence on the second/third instars' larvae of P. interpunctella with a 98.33% mortality rate under the laboratory conditions. There was a statistically significant difference in the mortality levels of the experimental group and control group (Pearson Chi-square, P: 0.02 < 0.05). It is considerable high effect when compared other Mattesia spp. (Alfazairy et al. 2019).
Mass rearing of both insects' P. interpunctella and E. kuehniella provides optimal conditions for reproduction and spread of the gregarine (Valigurova and Koudela 2006). The high infection of the Turkish strain of M. dispora in both laboratory and natural populations of P. interpunctella encouraged its mass production to be used in the biological control of P. interpunctella. There are some studies supporting this idea. Lord (2003) studied the alternative hosts that might be used for production of oocysts and revealed that G. mellonella larvae can serve as a medium for producing oocysts in larger quantities than the known small grain beetle hosts. Alfazairy et al. (2019) evaluated the potential of the Egyptian strain of the neogregarine, Mattesia sp., originally isolated from some stored grain insect pests in term of spore productivity, pathogenicity, and host range assays. According to those results, P. interpunctella could serve as a potential host for mass propagating the neogregarine pathogens. The present study confirms that P. interpunctella can serve as a medium for producing neogregarines in larger quantities and that M. dispora can be a natural suppressing factor of P. interpunctella population.

Conclusions
Neogregarines occur naturally in insect pest populations are highly pathogenic for them, therefore they have been considered as potential control agents against insect pests. Little is known about neogregarine infections as natural suppressing factor in pest populations. The Turkish strain of M. dispora is very common and widespread in the populations of P. interpunctella. Furthermore, it had a very high virulence against the P. interpunctella larvae. M. dispora can be an important natural suppressing protistan entomopathogen against P. interpunctella populations.