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.

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.

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.