Immunosuppressive effects of the limonoid azadirachtin, insights on a nongenotoxic stress botanical, in flesh flies.

The tetranortriterpenoid azadirachtin (Aza) is a well-known insect growth disruptor of plant origin. Although its actions on insects have been extensively studied; fragmentary reports are available from the immunological point of view. Therefore, in the present study, total (THC) and differential hemocyte counts (DHC), nodulation, phenoloxidase (PO) activity, immune-reactive lysozymes and inducible nitric oxide (NO) were assessed, as measures of immune responses, in Sarcophaga argyrostoma 3rd instars challenged individually with M. luteus or Aza, or in combination with both compared to the control larvae. THC was significantly declined after 12 h and 24 h of treatment with Aza. DHC varied considerably; in particular, plasmatocytes were significantly decreased after 36 h and 48 h of treatment with Aza; whereas granulocytes were significantly increased. Nodulation was significantly increased with the increase of time after all treatments. Challenging with M. luteus significantly increased the activity of PO in hemocytes and plasma; whereas such activity was significantly decreased after treatment with Aza or combined Aza and M. luteus. Treatment with Aza or M. luteus alone or in couple significantly increased lysozyme activity of fat body, hemocytes and plasma. However, challenging with M. luteus significantly increased NO concentration in the same tissues. A hypothetical model of Aza as a potential mutagen is presented. However, no genotoxic effect was observed through tracking apoptosis-associated changes in Aza-treated hemocytes via flow cytometry-based apoptosis detection. Our study suggests that the integration of Aza, as an eco-friendly pesticide, with bacterial biopesticides may be a successful approach for controlling insect pests.

Isolation, characterization, kinetics, and enzymatic and nonenzymatic microbicidal activities of a novel c-type lysozyme from plasma of Schistocerca gregaria (Orthoptera: Acrididae).

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 Ca(2+) or Mg(2+); 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.

Comparison of the hemolysis machinery in two evolutionarily distant blood-feeding arthropod vectors of human diseases.

Host blood protein digestion plays a pivotal role in the ontogeny and reproduction of hematophagous vectors. The gut of hematophagous arthropods stores and slowly digests host blood and represents the primary gateway for transmitted pathogens. The initial step in blood degradation is induced lysis of host red blood cells (hemolysis), which releases hemoglobin for subsequent processing by digestive proteolytic enzymes. The activity cycles and characteristics of hemolysis in vectors are poorly understood. Hence, we investigated hemolysis in two evolutionarily distant blood-feeding arthropods: The mosquito Culex pipiens and the soft tick Argas persicus, both of which are important human and veterinary disease vectors. Hemolysis in both species was cyclical after blood meal ingestion. Maximum digestion occurs under slightly alkaline conditions in females. Hemolytic activity appears to be of lipoid origin in C. pipiens and enzymatic activity (proteolytic) in A. persicus. We have assessed the effect of pH, incubation time, and temperature on hemolytic activity and the hemolysin. The susceptibility of red blood cells from different hosts to the hemolysin and the effect of metabolic inhibition of hemolytic activity were assessed. We conclude that in C. pipiens and A. persicus midgut hemolysins control the amplitude of blood lysis step to guarantee an efficient blood digestion.

Immune investigation of the honeybee Apis mellifera jemenitica broods: A step toward production of a bee-derived antibiotic against the American foulbrood.

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.

Assessment of oxidative stress and activities of antioxidant enzymes depicts the negative systemic effect of iron-containing fertilizers and plant phenolic compounds in the desert locust.

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 FeSO4 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 Fe2+ (as FeSO4) 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 Fe2+. K m 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 H2O2 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 FeSO4, and their degradation products. Our results demonstrated the stressful effects of L-DOPA and FeSO4, proving that iron-containing fertilizers are pollutants that can strongly affect S. gregaria.

Molecular characterization of a c-type lysozyme from the desert locust, Schistocerca gregaria (Orthoptera: Acrididae).

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.

Identification of digestive hydrolases in the larval midgut of the camel nasal bot fly Cephalopina titillator Clark (Diptera: Oestridae).

Activities of digestive hydrolases associated with midgut of the third instar larva of Cephalopina titillator were investigated. Based on the hydrolysis of synthetic substrates and optimum pH, it was found that C. titillator midgut contains trypsin-like (optimum pH, 9), chymotrypsin esterase-like (optimum pH, 8), carboxypeptidase A and B (optimum pH at 8.5 &7 respectively), alkaline- and acid-phosphatase (optimum pH at 9 & 5 respectively) and membrane bound leucine aminopeptidase (optimum pH, 8). An acid proteinase activity was detected, by the ability to hydrolyze acid denaturated haemoglobin; and it seems to be close to pepsin than cathepsin-like enzyme. It has a maximum activity at pH- 3.5. alpha-Glucosidase activity, and was also identified (optimum pH at 6) in the midgut, and seems to be membrane bound.