For herbivore insects, digesting can be somewhat challenging, as the defense mechanisms evolved by plants, including the release of phenolics like the non-protein amino acid L-3,4-dihydroxyphenylalanine (L-DOPA), can cause fitness costs. In addition, industrial and agricultural activities have elevated the amounts of iron that can be found in nature and more particularly FeSO that is used as fertilizer. Traces of iron can enhance the auto-oxidation of L-DOPA, in turn, generating reactive oxygen species (ROS) and consequently oxidative stress in insects. We examined the effects of the ion Fe (as FeSO) and L-DOPA on fifth instars of the desert locust Schistocerca gregaria. We measured the level of oxidative damage occurring to macromolecules (proteins and lipids) from midgut and thoracic tissues and assessed the activities of responsive antioxidant enzymes. Injected L-DOPA and redox-active metal iron generated ROS which caused oxidative damages to proteins and lipids to S. gregaria. The protein carbonyls and lipid peroxides present in tissue homogenates were elevated in treated insects. No synergism was observed when L-DOPA was co-injected with Fe. K values of superoxide dismutase (SOD), glutathione reductase (GR), and glutathione peroxidase (GPx) were 4.3, 2.6, and 4.0 mM in thoracic muscles and 5.00, 2.43, and 1.66 mM in whole midgut for SOD, GR, and GPx, respectively, and 8.3 and 3.43 M for catalase (CAT) in the two tissues, respectively. These results suggest higher affinities of GPx and CAT to HO in midgut than in muscles. The time-course changes in activities of antioxidant enzymes and amounts of protein carbonyls and lipid peroxides showed fluctuating patterns, suggesting complex interactions among macromolecules, L-DOPA and FeSO, and their degradation products. Our results demonstrated the stressful effects of L-DOPA and FeSO, proving that iron-containing fertilizers are pollutants that can strongly affect S. gregaria.

Lysozymes are bacteriolytic peptides that are implicated in the insect nonspecific innate immune responses. In this study, a full-length cDNA encoding a c-type lysozyme from Schistocerca gregaria (SgLys) has been cloned and characterized from the fat body of immune-challenged 5(th) instar. The deduced mature lysozyme is 119 amino acid residues in length, has a calculated molecular mass of 13.4 kDa and an isoelectric point (Ip) of 9.2. SgLys showed high identities with other insect lysozymes, ranging from 41.5% to 93.3% by BLASTp search in NCBI. Eukaryotic in vitro expression of the SgLys ORF (rSgLys) with an apparent molecular mass of ∼16 kDa under SDS-PAGE is close to the calculated molecular weight of the full-length protein. rSgLys displayed growth inhibitory activity against Gram-negative and Gram-positive bacteria. 3D structure modeling of SgLys, based on comparison with that of silkworm lysozyme, and sequence comparison with the helix-loop-helix (α-hairpin) structure of hen egg white lysozyme (HEWL) were employed to interpret the antibacterial potencies. Phylogenetic alignments indicate that SgLys aligns well with insect c-type lysozymes that expressed principally in fat body and hemocytes and whose role has been defined as immune-related. Western blot analysis showed that SgLys expression was highest at 6-12 h post-bacterial challenge and subsequently decreased with time. Transcriptional profiles of SgLys were determined by semi-quantitative RT-PCR analysis. SgLys transcript was upregulated at the highest level in fat body, hemocytes, salivary gland, thoracic muscles, and epidermal tissue. It was expressed in all developmental stages from egg to adult. These data indicate that SgLys is a predominant acute-phase protein that is expressed and upregulated upon immune challenge.

Abstract. The ability of biocontrol agents to overcome the immune defense of pests is a crucial issue. This is the first study of lysozyme activity as an inducible humoral component of the defense of Schistocerca gregaria, which depends on the recognition of the elicitor molecules of pathogens and not on epidermal wounding or a spiking effect. The level of lysozyme activity in fat body, haemocytes and haemolymph plasma of naïve and immunologically challenged 5 th instar S. gregaria was evaluated using the zone of inhibition test against Micrococcus lysodeikticus. Various Gram-positive and Gram-negative bacteria as well as peptidoglycans (PGN) and lipopolysacchrides (LPS) of bacterial cell walls induce and increase in the level of lysozyme activity. Escherichia coli induced an increase in the level of activity of lysozyme in the fat body, haemocytes and plasma, but not in mid gut epithelium, 6-12 h after an immunological challenge and then it decreased to the constitutive level after 72 h. This study revealed that in S. gregaria there is a constitutive and a bacteria-inducible level of lysozyme activity, which protects it against infection by both Gram-negative and Gram-positive bacteria.

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Nitric oxide (NO) plays various roles in insect immunity: as a cytotoxic component and as a signalling molecule; and immune-reactive lysozymes (IrLys) provide a first line of humoral immune functions against invading bacteria. Although there is considerable literature on eicosanoid and biogenic monoamine actions on insect immunity, there is no information on the role(s) of these chemicals in inducing NO and IrLys. We addressed this gap by challenging third instarSarcophaga(Liopygia)argyrostoma(Robineau-Desvoidy) with the Gram-positive bacteriumMicrococcus luteus. Here, we report that bacterial challenge induces elevation of NO and IrLys concentrations in haemocytes and in the fat body. The plasma pool content is comparatively low. Eicosanoid biosynthesis inhibitors (EBIs) lead to suppression of both NO and IrLys levels. Control larvae have low constitutive levels of NO and lysozyme concentrations. Octopamine (OA) elicits elevation of NO and IrLys concentrations. A similar effect is obtained by 5-hydroxytryptamine (5-HT) for NO. These data indicate immune-mediating roles of eicosanoids, OA and 5-HT in NO and IrLys activities.

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Keeping honeybees healthy is essential, as bees are not only important for honey production but also cross-pollination of agricultural and horticultural crops; therefore, bees have a significant economic impact worldwide. Recently, the lethal disease, the American foulbrood (AFB), caused great losses of honeybee and decline of global apiculture. Recent studies have focused on using natural insect-derived antibiotics to overcome recently emerged AFB-resistance to conventional antibiotics. In support of these studies, here we investigate the possibility of producing bee-derived anti-AFB antibiotics from an indigenous honeybee, Apis mellifera jemenitica. The immune responses of the third instar stage were first induced against the standards Micrococcus luteus and Escherichia coli compared with the indigenous Paenibacillus larvae (ksuPL5). Data indicated a strong immune response against M. luteus , E. coli and P. larvae 24 h post- P. larvae- injection as revealed by the detection of lysozyme-like, cecropin-like and prophenoloxidase (PO) activities in the plasma of P. larvae- injected third instars. Nodulation activity against injected P. larvae as early as 4 h and peaking 48 h post- P. larvae injection were observed. Potentially active anti- P. larvae immune peptide fractions purified by high-performance liquid chromatography (HPLC) showed significant in vivo therapeutic effects on P. larvae- infected first instars. Mass spectrophotometric analysis and Orbitrap measurements of P. larvae- injected plasma indicated the expression of PO (Mr: 80 kDa), beta-1,3-glucan-binding protein (Mr: 52 kDa) and serine protease 44 isoform X1 (Mr: 46 kDa). This suggests that one or all of these immune peptides contribute to significant survivorship of P. larvae -infected broods, and could be a valuable clue in the search for honeybee-derived anti-AFB natural therapeutic agents. Further molecular characterization and description of the functional roles of these predicted antimicrobial peptides from both broods and adult honeybee may enrich the arsenal of insect-derived antibiotics of therapeutic purposes.

A protein, designated as Sgl, showing a muramidase lytic activity to the cell wall of the Gram-positive bacterium Micrococcus lysodeikticus was isolated for the first time from plasma of Escherichia coli-immunized fifth instar Schistocerca gregaria. The isolated Sgl was detected as a single protein band, on both native- and SDS-PAGE, has a molecular weight of ∼15.7 kDa and an isoelectric point (pI) of ca 9.3 and its antiserum has specifically recognized its isolated form. Fifty-nine percentage of Sgl lytic activity was recovered in the isolated fractions and yielded ca 126-fold increase in specific activity than that of the crude. The partial N-terminal amino acid sequence of the Sgl has 55 and 40% maximum identity with Bombyx mori and Gallus gallus c-type lysozymes, respectively. The antibacterial activity against the Gram-positive and the Gram-negative bacteria were comparatively stronger than that of the hen egg white lysozyme (HEWL). The detected Sgl poration to the inner membrane that reach a maximum ability after 3 h was suggested to operate as a nonenzymatic mechanism for Gram-negative bacterial cell lysis, as tested in a permease-deficient E. coli, ML-35 strain. Sgl showed a maximal muramidase activity at pH 6.2, 30–50°C, and 0.05 M Ca2+ or Mg2+; and has a Km of 0.5 μg/ml and a Vmax of 0.518 with M. lysodeikticus as a substrate. The Sgl displayed a chitinase activity against chitin with a Km of 0.93 mg/ml and a Vmax of 1.63.