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Immune and defense mechanisms in representatives of Blattodea and Orthoptera: a review

Among insects orders, Blattodea and Orthoptera are characteristic with their high significance to human habitation, as posing medical and agricultural problems. Representatives of Blattodea have an important role as carriers of a number of infectious diseases in humans and animals, and are directly related to human life and activities. On the other hand Orthoptera are very significant as agricultural pests that cause great damage to plants. The study of the mechanisms of immune defense and the processes related to the response against pathogenic infections in these two orders is of interest in order to more fully clarify the possibilities for management and control of their populations. This review summarizes the information on the defense mechanisms (hemocytes, antimicrobial peptides, pathogen recognition, signaling patways, immune and antiviral responses) studied in representatives of these two orders. The list includes 30 species of cockroaches and termites and 59 orthopteran species, and focuses on species with medical significance (Periplaneta americana, Blattella germanica) and insect pests for agriculture such as Locusta migratoria and Schistocerca gregaria.


Introduction
Among the representatives of Blattodea, cockroaches are the most common pests in human dwellings. They are found in sewers, where they feed on waste and human feces and can spread a number of parasites and pathogens in the environment (Cotton et al. 2000, Pai et al. 2005. In addition, they contaminate food by leaving feces and bacteria that can cause food poisoning (Che Ghani et al. 1993), but can also carry bacteria, fungi and other pathogens in infected areas (Czajka et al. 2003, Kopanic 1994. Their nocturnal lifestyles and habitats make them ideal carriers of various infections (Allen 1987). For example, a study of Periplaneta americana (Linnaeus, 1758) shows that its individuals in a very high percentage carry a number of bacteria -Escherichia coli (86.7%), Proteus vulgaris (73.3%), Bacillus cereus (66.7%), Streptococcus faecalis (60%), Staphylococcus aureus (60%), Enterobacter cloacae (53.3%), Shigella spp. (33.3%), Serratia spp. (13.3%) and Staphylococcus epidermidis (6.7%) (Feizhaddad et al. 2012). In the cockroaches P. americana and Blattella germanica (Linnaeus, 1767), 25 species of bacteria have been isolated from hospital premises, of which 22 were gram-negative, and it was found that the cockroaches were more prone to carry pathogenic bacteria internally (84.3%) than on their body surface (64.1%) (Fakoorziba et al. 2010).
A number of parasites have been found on the surface or inside cockroaches, and some studies have shown that exposure to cockroach antigens may play an important role in asthma-related health problems (Montresor et al. 1998, Mott 1989). These insects, inhabiting various places and objects in people's houses (toilets, salons, kitchens and bedrooms) are potential reservoirs of a number of parasites of medical significance, transmitted through the surface of their bodies. These include mainly: cysts of amoebae (Entamoeba hystolitica), oocysts of coccidians (Cryptosporidium parvum, Cyclospora cayetenensis, Isospora belli), cysts of the protozoan Balantidium coli, eggs, larvae and adults of parasitic worms from the nematode group (Ascaris lumbricoides, Anchylostoma duodenale, Enterobius vermicularis, Trichuris trichura, Strongyloides stercoralis) (El-Sherbini & El-Sherbini 2011).
Most locusts also originate from pastures; grassland ecosystems are subject to agricultural expansion, urbanization, energy development and desertification, making them among the most endangered biomes on Earth (Hoekstra et al. 2005). For some grassland ecosystems and food plantations, the Moroccan grasshopper Dociostaurus maroccanus (Thunberg, 1815) or the red grasshopper Nomadacris septemfasciata (Audinet-Serville, 1883), this increase in anthropogenic change, together with modern control practices, has led to a reduction in outbreaks (Le Gall et al. 2019).
The current article summarizes species of both orders (Blattodea and Orthoptera) and the corresponding immune responses that are known in them, which is pertinent to the elucidation of the defense mechanisms and would facilitate a number of researchers in the field of biological control of insect pests. Significant species of cockroaches, termites, locusts, and crickets have been analized in more details.

Studies on the defense mechanisms of significant species of Blattodea and Orthoptera
Periplaneta americana P. americana is a species intensively studied for its immune mechanisms due to its importance as a carrier of a number of infectious diseases. Along with several other species, they are vectors of a number of pathogens (32 species of bacteria, including Salmonella and Shigella species, 15 species of fungi and molds, 7 helminths -intestinal parasites, 2 protozoa and 1 virus that are harmful to humans are carried in or on cockroaches and in their faeces (Mille & Peters 2004, Zarchi & Vatani 2009, Allotey et al. 2009, Akbari et al. 2015. Two species of cockroaches (B. germanica and Eublaberus posticus) have been studied in cultivationbased studies (Tachbele et al. 2006, Vahabi et al. 2007). Due to their significance as pests, studies on their immunity have justifiably been intensified. In a study of cockroach hemocytes, Scharrer (1972) reported that they contained a class of unusual cytoplasmic inclusion bodies which seem to undergo striking transformations in response to specific functional demands. Also noteworthy is that the capacity for the uptake of small particles by micropinocytosis is demonstrated by the localization of horseradish peroxidase activity at the cellular surface and within cytoplasmic vesicles. The author concluded that the diversity of structural appearances reflected a division of labor, while the many transitional features of hemocyte morphology favored the concept of functional flexibility of one basic cell type rather than a strict classification into distinctly separate cellular types.
When injecting a specific bee toxin, Karp & Rheins (1980) found a secondary immune response and that this humoral response of cockroaches to soluble protein toxins was specific and had the characteristics of immunological memory. Although insects have a simpler anatomy than vertebrates, the underlying molecular genetics may be as complex. In addition, vertebrate and invertebrate animals may be subject to equally complex, though different, environmental stresses (Karp 1990). Baines & Downer (1994) investigated the effects of 5-hydroxytryptamine, octopamine and dopamine on phagocytic activity and hemocyte nodule formation in cockroaches. Survival of cockroaches exposed to LD50 Staphylococcus aureus is increased in the presence of 5-HT and octopamine, while dopamine has no effect. Antagonistic and agonistic studies of cockroach survival support in vitro results, suggesting that both octopamine and 5-HT-sensitive receptors are found in cockroach hemocytes and are involved in the recovery of cockroaches from bacterial infection. Studies of hemocoele and hemocytes in treated cockroaches have shown at least two mechanisms by which 5-HT and octopamine can increase survival are by increasing phagocytosis and nodule formation. Shaban et al. (2010) investigated the cellular and humoral immune responses of adult American cockroaches, P. americana to the Egyptian entomopathogenic nematode Steinernema sp. Nematode injection reduced the total hemocyte count by 12 hours after injection, followed by an increase to the control level at 24 hours after injection. Phenoloxidase (PO) activity in the plasma of cockroaches injected with nematodes increased significantly at 12 hours postinjection, followed by a decrease at 24 hours post-injection. Both activities were significantly higher than those found in noninjected and water-injected P. americana plasma. The results suggest that the bacterial complex Steinernema has effective immunosuppressive mechanisms, leading to septicaemia and rapid death of the American cockroach. Mudoi et al. (2020) examined hemocytes in fungal infection with Beauveria bassiana, identifying changes in the dynamics of the groups of prohemocytes (PRs), plasma cells (PLs), granulocytes (GRs) and spherulocytes (SPs), their morphology. GRs, predominant hemocytes involved in cellmediated defense, responded to infection in different ways, including the formation of fine pseudopodia-like cytoplasmic extensions that accumulate together. Duarte et al. (2020) found that various stressful conditions such as starvation and dehydration do not affect the total number of hemocytes, but there are changes in their number depending on the type. In insects without food and water, the proportion of prohemocytes increases and plasma cells decrease. Hemocytes (Scharrer 1972); Hemocytes, Signaling (Lackie 1979 Hemocytes, Immune responses (Lackie et al. 1985); Immune responses (Dularay & Lackie 1987); Hemocytes, Immune responses (Karp 1990); Signaling, Immune responses (Tunaz & Stanley 2000); Immune responses (Hartman & Karp 1989); Immune responses (Faulhaber & Karp 1992); Signaling, Immune responses (Baines et al. 1992); Signaling, Immune responses (Baines & Downer 1994); Immune responses (Brown et al. 1994); Immune responses (Gritsai et al. 2004); Immune responses (Shaban et al. 2010); Hemocytes, Immune Responses (Mudoi et al. 2020); Immune responses (Duarte et al. 2020) Pseudacanthotermes spiniger AMPs (Lamberty et al. 2001)

Blattella germanica
The German cockroach, B. germanica, is a global pest that invades buildings, including homes, restaurants and hospitals, often maintained in unsanitary conditions. As a carrier of diseases and a producer of allergens, this species has major health and economic impact on humans. Factors contributing to the success of the German cockroach include its resistance to a wide range of insecticides, immunity to many pathogens and its ability, as an extremely universal omnivore, to survive on most food sources. B. germanica is a pest of public health worldwide that is difficult to control due to its strong reproductive capacity, adaptability and resistance to insecticides. A recently published genome shows that B. germanica has an extremely large number of genes encoding proteins (Harrison et al. 2018). Zhou et al. (2014) annotated and classified functionally in terms of BLAST, GO and KEGG, the genes putatively coding detoxification enzyme systems, insecticide targets, key components in systematic RNA interference, immunity and chemoreception pathways.
López-Uribe et al. (2016) in their study concluded that social insect species have developed behavioral immune defenses that reduce the risk of disease within the group, leading to lower immunity at the individual level. They suggest that insects living in large societies may rely more on behavioral mechanisms, such as hygiene behavior, than on immune function, to reduce the risk of disease transmission in breeding sites among emerging individuals. A significant negative effect on colony size on encapsulation response in individuals was found.
In a study by Harrison et al. (2018), the functions of the 93 significantly expanded gene families were studied in order to explain the successful development of B. germanica as a major pest despite adverse conditions. Large extensions have been found in gene families with functions related to the detoxification of insecticides and allelochemicals, protection against pathogens, digestion, sensory perception and gene regulation. This increase may allow B. germanica to develop multiple mechanisms of resistance to insecticides and pathogens, and allow for a broad, flexible diet, thus explaining its success in conditions of poor hygiene and repetitive chemical control. Silva et al. (2020) examining the German cockroach found that its defense systems require it to adapt to an unhealthy environment with an abundance of pathogenic microbes, in addition to the potential control of its symbiotic systems. To deal with this situation, four families of antimicrobial genes (defensins, termicins, drosomycins, and attacins) were expanded in its genome. Remarkably, a new family of genes (blatelicins) has recently emerged following the duplication and rapid evolution of the attacin gene, which now encodes a larger proteins with a long range of glutamines and glutamic acids. Screening for AMP gene expression in available transcriptional SR projects of B. germanica has shown that while some AMPs are expressed during almost all development, others are limited to shorter periods. Pan et al. (2020) highlighted new directions in the control of B. germanica, such as suppressing the cockroach population with Wolbachia or paratransgens and combining fungal insecticides with synergistic agents to increase insecticidal efficacy.
Order Blattodea also includes termites. Data show that various immune processes have been identified in 17 species of their representatives, e.g. production of antimicrobial peptides, hemocyte activities, immune responses, signaling [ Table 2].
Among Orthopteran species, most insect immunity studies have been performed on migratory locusts. Simonet et al. (2002) have cloned two serine protease inhibitor precursors in this species, contributing to the study of its immune signaling. Goldsworthy et al. (2003) demonstrated pathogen recognition via β-1,3 glucans as an early step in signaling, as opposed to virtually zero recognition of lipopolysaccharide (LPS) by the locust immune system. Similar findings have been demonstrated by Zheng & Xia (2012), which showed through RNA interference (RNAi) that β-1,3 glucan recognition protein (βGRP) is essential for resistance against fungal pathogens and opportunistic infections with insect gut symbionts.

Locusta migratoria
Antifungal immunity in L. migratoria is supplemented by a response known as "behavioral fever" whereby insects upregulate their body temperature in order to reduce their infection by a fungus, Metarhizium anisopliae (Ouedraogo et al. 2003). Antimicrobial peptides (AMPs) are the typical effectors of the insect immune system. A complete AMP transcriptome analysis of L. migratoria is lacking; nevertheless, Lv et al. (2016) have elucidated CSαβ defensins and defensin-like peptides in this species. Huybrechts & Coltura (2018) have pinpointed a role for angiotensin-converting enzyme (ACE) and ACE-inhibitors in fine-tuning the insect immune system by producing complementary AMPs. In a large-scale study on insect hemocytes, Zhang & Zhang (2021) have described hemocyte variations in 35 locust species, including L. migratoria. Other Orthopteran species which are objects of insect immunity studies include, notably, the desert locust Schistocerca gregaria (Forsskål, 1775) and the house cricket Acheta domesticus (Linnaeus, 1758).

Schistocerca gregaria
The desert locust, S. gregaria, has been investigated to a greater depth in its role as a pest and potential target for biological control. Early on, Lackie et al. (1985) elucidated hemocyte profiles and encapsulation responses. Gillespie et al. (2000) have pinpointed the susceptibility of S. gregaria to fungal infection by Metarhizium anisopliae. While desert locusts also exhibit the protective response known as "behavioral fever", Bundey et al. (2003) demonstrated that it's an integral part of the insect's immune system by showing that it can be suppressed by the corticosteroid dexamethasone. Tounou et al. (2008) demonstrated that the microsporidium Nosema (Paranosema) locustae Canning, 1953 is an efficient control agent for S. gregaria populations, limiting the number of individuals that reach adulthood and discovered mostly synergistic effects during co-infection of S. gregaria with M. anisopliae. While Simonet et al. (2005) reported the expression of pacifastin-like peptide precursors in desert locusts, surprisingly little is known about the transcriptome of S. gregaria under conditions of stress and infection and the effectors of its immune system. Together with an apparent lack of studies of locust immunity against viral pathogens, this represents a large gap in the current knowledge of immunity in this species. It is projected that, due to the significant role of S. gregaria as pest in Sub-Saharan Africa, its role in future studies of insect immunity will increase.

Acheta domesticus
The house cricket A. domesticus is popular among insect breeders around the world specifically due to its high nutritional value and suitability as food source for amphibian and reptilian exotic pets. Nevertheless, its prevalence as live food source has been declining recently due to its susceptibility to the Cricket Paralysis Virus (CrPV), which causes 95% mortality in infected cricket species (Plus & Scotti, 1984). Early research with crickets focused on pathogen-induced behavioural fever (Adamo 1998). Ardia et al. (2012) have found tradeoffs between induced immune responses in A. domesticus and metabolic rate/antimicrobial activity. Sorrell & Killian (2020) investigated the influence of RNA interference (RNAi) of the fragile X mental retardation gene in house crickets, concluding that its deletion damages the immune system in male, but not female crickets. Reginald et al. (2021) studied the immune responses of A. domesticus to injections of pathogenic Escherichia coli K1 bacteria, elucidating signaling via prophenoloxidase (ProPO) and responses, associated with hemocyte biogenesis. Overall, A. domesticus has served as a model organism in some insect immunity studies, but research has been sporadic, with little systematic understanding of this species immune system, especially in response to fungal and viral pathogens.

Conclusion
Both orders of insects belong to the group of hemimetabolan insects and although they differ from a biological and ecological point of view, they are important for their role as vectors of a number of infectious diseases (Blattodea) as well as enemies with significant economic importance in the field of agriculture (Orthoptera). In this regard, the study of the defense mechanisms of these insects is important for a more in-depth study of the possibilities of control when their population density is high. The processes of innate immunity established so far (hemocytes activities, antimicrobial peptides, pathogen recognition, signaling pathways, immune and antiviral responses) are important and reveal new data and facts, which would lead to gaps in knowledge regarding the effects of various groups of pathogens on them and the registered insect physiological protective responses.