Examination of insect immunity helps to understand why insects are resistant or susceptible to a variety of harmful factors of environment. One of the most interesting point is understanding that irritation of insect immune system by one kind of agents cause altered sensitivity to others. This leads to explanation and maybe prediction of dynamics of development of mixed infections and of insect pathogenesis under stress conditions. Host immune responses, underlying parasite-host interactions of microsporidia and insects, are poorly studied. In our lab, we study various aspects of host immune reactions in three microsporidia-insect parasite-host systems: Gryllus bimaculatus - Paranosema (=Nosema) grylli; Locusta migratoria - P. locustae; and Galleria mellonella - Vairimorpha ephestiae.

Major cellular reactions of crickets and locusts to microsporidian infection are phagocytosis of parasite spores, incapsulation and melanization of infected cells. During early stage of disease, no signs of cellular responses are found in cricket tissues. At this period, proliferate parasite stages reside inside the host cells, avoiding non-self recognition by host haemolymph. Melanized nodules and incapsulated cells within infected fat bodies and lymph glands of crickets are associated with mature Paranosema grylli spores. At the acute stage of disease, when mass sporogony of microsporidia occurs, up to 90% crickets manifest melanization of infected tissues. Incapsulation of infected cells is mediated by plasmatocytes and granulocytes, the major cell types of cricket haemocytes (Fig. 1) (Sokolova et al., 2000). Ruptured infected cells release large amount of spores into the body cavity, were phagocytosis takes place (Fig. 2). Spores uptaken by phagocytes both in vivo and in vitro undergo acidification and degradation within the phagosomes. Some of the incapsulated and phagocytized spores are exfilamented, thus escaping elimination by host immune system.
In locusts, cellular reactions in tissues massively infected with mature spores of P. locustae are very weak or absent even at the final stage of disease. We suppose that this fast developing and highly aggressive parasite with a broad host range is able to suppress host immune responses (Tokarev et al., 2004a).

Fig. 1. Major types of cricket haemocytes: prohaemocytes (1), plasmatocytes (2) and granulocytes (3).		Fig. 2. Phagocytosis of microsporidian spores by cricket haemocytes: spore adhesion to the cells (left); a semithin section through a haemocyte with ingested spores (right).

Melanization, mediated by phenoloxidase (PO) system, is a principal defense reaction of insect haemolymph against invading pathogens, and many parasites exploit diverse strategies to suppress or avoid action of POs. Involvement of PO in interactions of microsporidia with insect immune system is poorly studied. We have demonstated previously, that microsporidia P. grylli suppress PO in haemocytes of G. bimaculatus at the acute stage of microsporidiosis (Sokolova et al., 1999, 2000). In the present study it was found that:
(1) Microsporidian spores suppress PO activity both in plasma and in haemocytes. Whole haemolymph samples of G. bimaculatus and G. mellonella did not melanize, and levels of PO-positive haemocytes were decreased at the phase of microsporidian spore producion, as compared to naive insects. PO activity of haemocytes was also suppressed after injection of spores into insect body cavity, as well as after cooincubation with spores in vitro.
(2) Melanin granules in infected tissues of crickets and locusts were associated with microsporidian spores, and never with intracellular stages (Fig. 3).
(3) Melanized nodules in G. bimaculatus and L. migratoria contained enlarged spores with aberrant nuclear apparatus (Fig. 4). Quote of these spore inside nodules was 6-10 times higher than outside.
The obtained results showed that suppression of PO activity took place in all studied systems, though it may vary in extent and patterns, and bring some evidence that melanin deposits are toxic for microsporidia development and may lead to formation of teratospores. Ability to suppress PO synthetic pathway might be considered as an adaptation of insect microsporidia to survive inside the host (Tokarev et al., 2004a).

Fig. 3. Black melanin granules in cricket fat body at the acute microsporidosis		Fig. 4. Teratospores of Paranosema grylli, stained with DAPI (left), showing abberant nuclear apparatus, and of P. locustae, stained with Calcoflour (right), showing irregular shape and size compared to normal (N) spores

Insect pathology is a fascinating world where interests of several disciplines meet: entomology, parasitology, microbiology, ecology etc., and applied sciences, such as biological control of pests and disease vectors, have a lot to do with it as well. Alongside with microsporidia research, which has the utmost priority in our scientific group, other pathogens of insects are also in the scope of our attention, both fundamental and applied.

Development of biological control measures of locusts and grasshoppers is one of most priority and fast developing directions in modern IPM programs. The most important from this point of view are entomopathogenic hyphomycetes (Deuteromycota, Hyphomycetes). Due to a high level of reproduction of Calliptamus italicus and Locusta migratoria on the south of Russia, we studied activities of fungi against locusts under lab and field conditions, together with the Fungi research group (Dr. G.R. Lednev, Ph.D. student M.V. Levchenko) from our institute.
Several strains of Beauveria bassiana (Fig. 7) and one of Metarhizium anisopliae (Fig. 8), isolated recently, are being studied now, with respect to their virulence, toxigenicity, character of in vitro growth and modes of application against locusts. We concentrate mainly on the most effective strain of M. anisopliae, and factors affecting its efficacy (weather conditions, mode of application and exposure time, in vivo passaging of the fungus, host immunity suppression) are examined in detail (Tokarev et al., 2004b).

Fig. 7. Beauveria bassiana grown on agar plate (left) and on its locust host (right). Photos from Dr. G. Lednev		Fig. 8. Metarhizium anisopliae grown on agar plate (left) and on its locust host (right). Photos from Dr. G. Lednev

Infections caused by more than one pathogen simultaneously are of particular interest, as in nature, where many types of organisms meet, probability of pathogenesis with mixed ethiology is quite high. It is also important to know how would reactions of an insect population to implication of chemical and biological control agents differ under influence of naturally occuring or introduced parasites. Besides microsporidia, in our lab cultures of orthopteran insects there's a set of other pathogens: coccidia (Fig. 9), gregarines and viruses in crickets; gregarines and amoebae (Fig. 10) in locusts. Observations of dynamics of development of these parasites are done during experimental microsporidiosis to elucidate role of microsporidia in mixed infections, as the latter suppress host immunity system. Moreover, in bioaasays with microsporidia and fungi (Beauveria bassiana and Metarhizium anisopliae), that have potential in locust biocontrol, we study alterations of insect sensitivity to application of fungi and their toxins at certain phases of microsporidiosis .

Fig. 9. Coccidian Adelina grylli: stages of shizont maturation (left); sporulated oocyst (right). Photos from Dr. J. Sokolova		Fig. 10. Cysts of Malamoeba locustae in locust tissues

Indentification of pathogens in field populations and lab cultures of insects is necessary for a wide spectre of reasons, including right interpretation of results of physiological studies, survey of prevalence and distribution of parasites in host populations, search for new species of protists and prediction of insect pest densities changes under pressure of pathogens. Main methods for diagnosis of pathogens in insects are routine light microscopical observation combined with histochemical staining. Staining with fluorescent probes for nuclei and chitin-like compounds (see, for example, Fig. 4) are employed for more precise recognition of parasitic stages and some characteristic features of their cells. Methods for rapid determination of viability of invasive stages of microsporidians and other pathogens are also being tested and compared in our lab.