Nitric oxide synthase activity in tissues of the blowfly Chrysomya megacephala (Fabricius, 1794)

A. C. Faraldo¹, A. Sá-Nunes², L. H. Faccioli², E. A. Del Bel³, And E. Lello⁴

¹ Universidade Federal Rural de Pernambuco, Unidade Acadêmica de Garanhuns, UFRPE/UAG, Garanhuns, Pernambuco, Brazil.
² Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, USP, Ribeirão Preto, São Paulo, Brazil.
³ Departamento de Morfologia, Estomatologia e Fisiologia, Faculdade de Odontologia de Ribeirão Preto, USP, Ribeirão Preto, São Paulo, Brazil.
⁴ Departamento de Morfologia, Instituto de Biociências, UNESP, Botucatu, São Paulo, Brazil.

Address correspondence to: Dr.  Ana Carolina Faraldo. Universidade Federal Rural de Pernambuco, Unidade Academica de Garanhuns, Avenida Bom Pastor, s/n, Mundaú, 55.292-901, Garanhuns, PE, BRAZIL. Fax: +55 87 37613233. E-mail: acfaraldo@yahoo.com

ABSTRACT:  Although insects lack the adaptive immune response of the mammalians, they manifest effective innate immune responses, which include both cellular and humoral components. Cellular responses are mediated by hemocytes, and humoral responses include the activation of proteolytic cascades that initiate many events, including NO production. In mammals, nitric oxide synthases (NOSs) are also present in the endothelium, the brain, the adrenal glands, and the platelets. Studies on the distribution of NO-producing systems in invertebrates have revealed functional similarities between NOS in this group and vertebrates. We attempted to localize NOS activity in tissues of naïve (UIL), yeast-injected (YIL), and saline-injected (SIL) larvae of the blowfly Chrysomya megacephala, using the NADPH diaphorase technique. Our findings revealed similar levels of NOS activity in muscle, fat body, Malpighian tubule, gut, and brain, suggesting that NO synthesis may not be involved in the immune response of these larval systems. These results were compared to many studies that recorded the involvement of NO in various physiological functions of insects.

Key words: NO synthase; Nitric oxide; NADPH-diaphorase; Chrysomya megacephala; Blowfly; Tissues.

Introduction

     Calliphorid blowflies have substantial medical and veterinary importance because they produce myiasis in humans and other animals and may be mechanical vectors of enteric pathogens and parasites (Furlanetto et al., 1984; Wells, 1991). Because both immature and adult stages are the primary invertebrate consumers of decomposing animal organic matter (such as carrion), blowflies can play a valuable role in forensic medicine, helping to determine time, manner, and place of death (Catts and Goff, 1992; Wells and Kurahashi, 1994). Chrysomya is an abundant blowfly genus that is native of Africa. This genus was established in South America in 1975 (Guimarães  et al., 1978) and its distribution reaches also North America (Greenberg, 1988). These insects are holometabolous, and the larvae go through three instars when they live in or feed on recently dead animals or carrion. During this period, they feed on a substrate with a high concentration of micro-organisms. When they disperse outside their original food source to search for a suitable site for pupariation, they are susceptible to parasitism and physical stress. Consequently, these larvae must possess an extremely efficient immune system. Therefore, they may be an excellent model for studying insect defense mechanisms.
     Although lacking the components that characterize the acquired immunity systems of vertebrates, insects are known to possess efficient mechanisms for combating pathogens by building up defense responses, which exhibit striking parallels with those of the innate immunity of vertebrates.These innate immune systems include both cellular and humoral elements. The cellular components of insect immunity, called hemocytes, are able to phagocytose, nodulate, and encapsulate. Phagocytosis is the first barrier against foreign bodies, and it has been described in the hemolymph of many insect species to fight against biological (Ratcliffe and Rowley, 1979; Ratcliffe  et al., 1985; Götz and Boman, 1985; Ratcliffe, 1986) and non-biological agents (Wiesner, 1991, 1992; Slovák  et al., 1991; Faraldo and Lello, 2003). If a considerable number of elements invade the hemocoel, the elements are isolated by hemocyte aggregation and form nodules that may or may not be melanized (Ratcliffe and Rowley, 1979; Lackie, 1980). The encapsulation by hemocytes occurs when the foreign body is too large to be phagocytosed. Many studies have revealed that both humoral and cellular factors contribute to this encapsulation reaction (Götz and Vey, 1987; Götz et al., 1987; Rizki and Rizki, 1987; Faraldo et al., 2005). Humoral immunity involves (a) the induction of proteolytic cascades that cause hemolymph coagulation (Muta and Iwanaga, 1996) and localized melanization (Nappi and Vass, 1993; Carton and Nappi, 1997); (b) the oxygen-dependent mechanisms that include the synthesis of lysozyme, proteolytic, and hydrolytic enzymes, and antimicrobial peptides (Meister et al., 1997); and finally (c) the generation of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) (Nappi et al., 1995; Nappi and Vass, 1998; Luckhart et al., 1998; Nappi et al., 2000; Nappi and Ottaviani, 2000; Whitten et al., 2001). In mammalian immunity, among the nitrogen radicals, nitric oxide (NO) is best known for its anti-microbial activity by killing microorganisms by nitrosylation, nitration, and oxidation of essential microbial components (DNA, lipids, and proteins) (Fang, 1977).
     The production of NO results from ligand-receptor interactions and is generated among citrulline during the NOS-catalyzed hydrolysis of L-arginine. These enzymes require NADPH as co-factor and are inhibited by some analogues of  L-arginine. Three isoforms of NOS have been described in vertebrates: two are constitutive (cNOS) and one is inducible (iNOS). In mammals, cNOSs are present in the tissues of the endothelium, the brain, the adrenal glands, and in platelets (Moncada et al., 1991). The iNOS, synthesized in response to cytokines and inflammatory mediators, has been identified in macrophages, hepatocytes, vascular smooth muscle, endothelial cells and neutrophils (Moncada et al., 1991; Rimele et al., 1991). NOSs were originally identified in mammalian tissues. However, NOS genes were cloned from some insect species (Drosophila melanogaster, Anopheles stephensi, Anopheles gambiae, Rhodnius prolixus) and the amino-acid sequences deduced exhibited similarities with those of Ca²⁺/calmodulin-dependent NOSs from mammals (Regulski and  Tully, 1995; Luckhart  et al., 1998; Luckhart and Rosenberg, 1999; Dimopoulos et al., 1998; Yuda et al., 1996). Martínez (1995) has showed evidence for NOS activity in invertebrate tissues. However, there are few studies that relate NOS activity to immune responses in insect tissues (Ribeiro and Nussenzveig, 1993; Luckhart et al., 1998; Dimopoulos et al., 1998; Whitten et al., 2001; Foley and O'Farrel, 2003; Faraldo et al., 2003).
     To elucidate the relation between NO generation in insect tissues and immune mechanisms, this study aims to identify tecidual NOS activity among naïve larvae, yeasts inoculated-larvae, and saline injected-larvae of Chrysomya megacephala and to outline comparisons with other insect orders.

Materials and methods

Insects

     Newly hatched larvae of Chrysomya megacephala were obtained from adults kept at constant temperature (25°C), and raised in vials containing minced meat.The postfeeding larvae were removed from the vials, washed in 10% clorhexidine solution, immersed in 10% sodium hypochloride (NaClO) solution for 10 minutes, and washed in distilled water.

Microorganisms

     Saccharomyces cerevisae yeasts were used in all experiments. C. megacephala reactions against these microorganisms were the model used to study the immune responses in this insect.
     S. cerevisae yeasts (Fleischmann^®) were suspended in saline solution for insects (SSI: 154mM NaCl, 126mM KCl, 7.2mM CaCl₂, 0.24mM NaHCO₃, pH 7.0) and stored at 4°C. Before each experiment, the yeasts were resuspended in the solution mentioned above, and the suspension was adjusted to 10⁵ yeast/ml. The volume of yeast suspension injected in each larva was approximately 20 mL.

Localization of nitric oxide synthase (NOS)

     NOS was localized  in situ among naïve larvae (uninjected, UIL), yeasts-injected larvae (YIL), and larvae injected with SSI (SIL). Samples were taken from injected-groups 24 h post-injection, once our previous experiments showed there was increased NO production in the blowfly hemolymph at this time after yeast injection (Faraldo  et al., 2005). Because NOSs have NADPH diaphorase activity, a histochemical technique for NADPH diaphorase was used to locate the enzyme.

Nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase technique

     Injected or uninjected larvae (YIL, SIL, and UIL) were washed in phosphate-buffered saline (PBS, 0.01 M NaCL and 10 mM NaPO4, pH 8.4), fixed in 4% paraformaldehyde (PFA) for 24 h and incubated in PFA + 15% sucrose or PBS + 15% sucrose for 24 h.
     The assays were performed in larval sections. All the samples were frozen at -70°C, embedded in Tissue Tek^® (Sakura Finetek) at the same temperature, and 16 mm-cryo sectioned in a cryostat Leica CM 1850. The sections were placed in slides and incubated in 0.2 M PBS with 1 mM NADPH, 1 mg/ml nitroblue tetrazolium, 3%Triton X-100, and distilled water at 37°C. After 90 min, the sections were examined for specific staining by the diaphorase technique under light microscopy and documented in a Photomicroscope (Zeiss II).

Results

     The NADPH diaphorase staining method revealed NOS activity in tissues of the muscles (M), the fat body (FB), the Malpighian tubules, the gut (G) and the brain (B) of naïve (UIL), yeast-injected (YIL), and saline-injected (SIL) larvae of Chrysomya megacephala (Figs.1-5). These larval tissues showed similar staining patterns among the three groups treatments.

FIGURE 1-5.  Cryosections of  C.  megacephala larvae.  NADPH-diaphorase staining technique, which indicates NOS activity was used. 1. Brain (B) and muscle (M) of naïve larva (uninjected, UIL) showing positive reaction. Arrow: larval cuticle; T: trachea. Bar: 20 mm. 2. Stained Malpighian tubules (arrow) of UIL at high magnification. M: muscle; T: trachea. Bar: 10 mm. 3. Muscle (M), gut (G) and fat body (FB) of yeast-injected larvae (YIL) stained by the NADPH-diaphorase technique. Arrow: cuticle. Bar: 20 mm. 4. Stained gut (G) and fat body (FB) of YIL at high magnification. Bar: 5 mm. 5. Gut (G) and fat body (FB) of saline-injected larvae showing positive reaction. Bar: 5 mm.

Discussion

     The NADPH diaphorase histochemical technique was applied to locate NOS activity in UIL,YIL and SIL tissues to attempt to link this activity to immune mechanisms. In this reaction, the oxidation of NADPH by diaphorase is coupled with the reduction of nitroblue tetrazolium (NBT), which precipitates as dark blue formazan and indicates the location of NOS, the enzyme responsible for biosynthesis of nitric oxide (NO) (Dawson et al., 1991). Presumably, the NBT-formazan precipitation observed here does not correspond to NO generation in response to the yeast inoculation, but to the activity of NOSs probably involved in distinct physiological functions of the blowfly tissues.
     NOS activity has been demonstrated in invertebrate tissues (Martínez, 1995; Elphicket al., 1993; Stefano and Ottaviani, 2002). In recent years, more evidence for insect NOSs has been reported (Davies et al., 1997; Müller, 1997; Luckhart et al., 1998; Chiang et al., 2000;Whitten et al., 2001; Ott and Elphick, 2003); however, there has not been data indicating NOS activity in insect muscles. In his review, Martínez (1995) reported the presence of diaphorase reaction in muscle cells of a mollusk species. He also inferred that NOS activation in muscles of more evolved invertebrates might be regulated by the nervous system. Wildemann and Bicker (1999) investigated an NO signaling system at the neuromuscular junction (NMJ) of the Drosophila melanogaster larvae. By combining immunocytochemical and exocytosis images, these authors demonstrated the involvement of NO in the regulation of synaptic vesicle release at the NMJ level in this dipteran. Our results suggest the NOS activity located in muscle tissue of C. megacephala might be located at the nerve ending, where NO might play a role in neurotransmission, as it does in  Drosophila (Wildemann and Bicker, 1999) and mammals (Moncada et al., 1991).
     Otherwise, NOS activity has been described in insect fat body (FB) and Malpighian tubules (MT) (Martínez, 1995; Choi et al., 1995; Davies et al., 1997; MacPherson, 2001). These organs have similar functions to those attributed to the mammalian liver and kidney, respectively. Choi et al. (1995) identifies distinct types of NOSs in the FB and MT of the silkworm Bombyx mori. According to the authors, the major FB NOS is inducible by lipopolysaccharade (LPS), such as iNOSs from mammal hepatocytes (Moncada  et al., 1991). The two NOSs found in MT were constitutive. However, Choi et al. (1995) strongly suggest that these NOSs are related to the insect metamorphosis. By contrast, it is proposed that the activation of NOS in MT of Drosophila is involved in the stimulation of fluid secretion (Davies et al., 1997).A similar function is described for NOS for the mammalian kidney (Springall et al., 1992), which suggests that this dipteran's MT NOS is also involved in the regulation of the excretory system. NOSs activities detected in both FB and MT of  C. megacephala larvae occurred in all the groups and may represent constitutive NOSs. Nonetheless, experimental evidence is needed to establish whether detected NOSs are involved in the control of these systems or in the metamorphosis, as described for locusts (Choi  et al., 1995).
     NOS activity in the insect gut has been previously described (Luckhart  et al., 1998; Dimopoulos  et al., 1998; Hao  et al., 2003). Using diaphorase staining, Luckhart et al. (1998) have already demonstrated the presence of NOS in the midgut of the malaria vector Anopheles stephensi. This inducible NOS activity is higher in Plasmodium-infected mosquitoes and is able to limit parasite development (Luckhart et al., 1998; Lim et al., 2005). In a similar way, there is recent evidence of increased NOS activity at the foregut/midgut junction (proventriculus) in tsetse fly, upon microbial challenge (Hao et al., 2003). We examined sections of naïve, yeast-injected, and saline-injected larvae of C. megacephala, and in all the groups, the use of the NADPH diaphorase technique revealed NOS activity in the gut. This NOS activity could be due to the elevated concentration of microorganisms, commonly present in the insect meals, which suggests that NOS may also exert an anti-microbial effect in the  C. megacephala gut. Further studies are required to determine whether NOS activity in the blowfly larval gut is inducible or constitutive.
     Finally, in most of the invertebrates studied, NOS appear to be involved in neurotransmission in both the central and the peripheral nervous systems (Martínez, 1995). The brain in insects, as well as in vertebrates (Salter et al., 1991), have been described to posses the highest activity of constitutive NOS (Müller, 1997). Using NADPH diaphorase staining, Chiang et al. (2000) detected cNOS activity in the cerebral ganglion of Periplaneta americana. Subsequently, they demonstrated that the high cNOS activity expressed in cockroach corpora allata (CA) was also expressed in other insects, such as the house cricket, a lepidopteran species, and the fruit fly. The authors assumed the occurrence of cNOS in the CA of most, if not all insects, and suggested that CA releases NO as a messenger molecule in these arthropods. Activity of cNOS was also identified in several parts of the Schistocerca gregaria brain (Elphick et al., 1995). Based on the particular abundance of cNOS in the olfactory system, NO involvement in the locust olfaction was suggested.
     To date, in addition to the olfactory signal transdution, NO generation seems to be implicated in memory formation in the honeybee Apis mellifera, which is a finding which provides remarkable parallels with findings in vertebrates (Menzel, 2001). In a recent review, Bicker (2005) indicated that NOS affects the wiring of insect nervous systems by regulating cell motility. Our histochemical essays revealed NOS activity in the central region of the C. megacephala larval brain. Interestingly, the stained portion seems to correspond to the central complex of adults, which contains the olfactory center. Olfaction is thought to be essential for larval nutrition (Hancock and Foster, 1997) and, consequently, for successfully reaching the adult stage.
     The main objective of this study was to investigate NOS activity in blowfly tissue in response to yeast inoculation by the NADPH diaphorase method. Our findings revealed NOS staining levels in the tissues of the muscle, the fat body, the Malpighian tubules, the gut, and the brain, were similar before and after the challenge. This is the first study that shows the synthesis of NO in larval tissues of the blowfly  Chrysomya megacephala. Many studies are emerging that register the implication of NO in various physiological functions of insects, and functional role of NOS in these blowfly systems certainly warrants consideration in future investigations.

Acknowledgments

The authors are grateful to Renata da Silva Ferreira and Célia Aparecida da Silva for technical assistance. This work was supported by Grant 00/00718-9 from FAPESP (Fundação de Apoio à Pesquisa do Estado de São Paulo).

References

1. Bicker G (2005). Stop and go with NO: Nitric oxide as a regulator of cell motility in simple brains. BioEssays 27: 495-505.         [ Links ]

2. Carton Y, Nappi AJ (1997). Drosophila cellular immunity against parasitoids. Parasitol. Today 13: 218-227.         [ Links ]

3. Catts EP, Goff ML (1992). Forensic entomology in criminal investigations. Annu Rev Entomol. 37: 253-272.         [ Links ]

4. Chiang A-S, Wen C-J, Lin C-Y, Yeh C-H (2000). Nadph-diaphorase activity in corpus allatum cells of the cockroach, Diploptera punctata. Insect Biochem Mol Biol. 30: 747-753.         [ Links ]

5. Choi SK, Choi HK, Okuda KK, Taniai K, Kato Y, Yamamoto M, Chowdhury S, Xu J, Miyanoshita A, Debnath CN, Asaoka A, Yamakawa M (1995). Occurrence of novel types of nitric oxide synthase in the silkworm, Bombyx mori. Biochem Biophys Res Com. 207(1): 452-459.         [ Links ]

6. Davies SA, Stewart EJ, Huesmann GR, Skaer NJ, Maddrell SH, Tublit NJ, Dow JA (1997). Neuropeptide stimulation of the nitric oxide signaling pathway Drosophila melanogaster Malpighian tubules. Am J Phisiol. 273(2Pt2): R823-R827.         [ Links ]

7. Dawson TM, Bredt DS, Fotuhi M, Hwang PM, Snyder SH (1991). Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues. Proc Natl Acad Sci. USA 88: 7797-7801.         [ Links ]

8. Dimopoulos G, Seeley D, Wolf A, Kafatos FC (1998). Malaria infection of the mosquito Anopheles gambiae activates immuneresponsive genes during critical transition stages of the parasite life cycle. EMBO J. 17: 6115-6123.         [ Links ]

9. Elphick MR, Green IC, O'Shea M (1993). Nitric oxide synthesis and action in an invertebrate brain. Brain Res. 619: 344-346.         [ Links ]

10. Fang FC (1977). Mechanisms of nitric oxide-related antimicrobial activity. J Clin Invest. 100: S43-S50.         [ Links ]

11. Faraldo AC, Lello E (2003). Defense reactions of  Dermatobia hominis (Diptera: Cuterebridae) larval hemocytes. Biocell 27(2): 197-203.         [ Links ]

12. Faraldo AC, Sá-Nunes A, DelBel EA, Faccioli LH, Lello E (2005). Nitric oxide production in  Chrysomya megacephala hemolymph after yeast inoculation. Nitric Oxide: Biol Chem. 13(3): 196-203.         [ Links ]

13. Faraldo AC, Gregório EA, Lello E (2005). Nodule formation in blowfly hemolymph after yeast inoculation. Brazilian J Morphol Sci. S134.         [ Links ]

14. Foley E, O'Farrel PH (2003). Nitric oxide contributes to induction of innate immune responses to gram-negative bacteria in Drosophila. Genes Devel. 17: 115-125.         [ Links ]

15. Furlanetto S M, Campos MLC, Harsi CM (1994). Microorganismos enteropatogênicos em moscas africanas pertencentes ao gênero Chrysomya  (Diptera: Calliphoridae) no Brasil. Revta Microbiol. 15: 170-174.         [ Links ]

16. Götz P, Boman HG (1985). Insect immunity. In: Comprehensive insect physiology, biochemistry and pharmacology. Kertut GA, Gilbert LI (Ed.). Oxford: Pergamon Press. pp.454-485.         [ Links ]

17. Götz P, Enderlein G, Roettgen I (1987). Immune reactions of Chiromonus larvae (Insecta: Diptera) against bacteria. J Insect Physiol. 3: 993-1004.         [ Links ]

18. Götz P,  Vey  A (1987). Humoral encapsulation in insect. In: Hemocytic and humoral immunity in arthropods. Gupta AP (Ed). New York: Wiley Interscience. pp. 407-430.         [ Links ]

19. Greenberg B (1988). Chrysomya megacephala (F.) (Diptera: Calliphoridae) collected in North America and notes on Chrysomya species present in the New World. J Med Entomol. 25: 199-200.         [ Links ]

20. Guimarães JH, Prado AP, Linhares AX (1978). Three newly introduced blowfly species in Southern Brazil (Diptera: Calliphoridae). Revta Bras Ent. 22: 53-60.         [ Links ]

21. Hancock RG, Foster WA (1997). Larval and adult nutrition effects on blood/nectar choice of Culex nigripalpus mosquitoes. Med Vet Entomol. 11(2): 112-122.         [ Links ]

22. Hao Z, Kasumba I, Aksoy S (2003). Proventriculus (cardia) plays a crucial role in immunity in tsetse fly (Diptera: Glossinidae). Insect Biochem. Mol Biol. 33(11): 1155-1164.         [ Links ]

23. Lackie AM (1980). Invertebrate immunity. Parasitology 20: 393-412.         [ Links ]

24. Lim J, Gowda DC, Krishnegowda G, Luckhart S (2005). Induction of nitric oxide synthase in Anopheles stephensi by Plasmodium falciparum: mechanism of signaling and the role of parasite glycosylphosphatidylinositols. Infect Immun. 73(5): 2778-2789.         [ Links ]

25. Luckhart S, Vodovotz Y, Cui L, Rosenberg R (1998). The mosquito Anopheles stephensi limits malaria parasite development with inducible synthesis of nitric oxide. Proc Natl Acad Sci. USA 95: 5700-705.         [ Links ]

26. Luckhart S, Rosenberg R (1999). Gene structure and polymorphism of an invertebrate nitric oxide synthase gene. Gene 232: 25-34.         [ Links ]

27. MacPherson MR, Pollock VP, Broderick KE, Kean L, O'Connel FC, Dow JAT, Davies SA (2001). Model organisms: new insights into ion channel and transporter function L-type calcium channels regulate epithelial fluid transport in Drosophila melanogaster. Am J Physiol Cell Physiol. 380: C395-C407.         [ Links ]

28. Martínez  A (1995). Nitric oxide synthase in invertebrates. Histochem J. 27: 770-776.         [ Links ]

29. Meister M, Lemaitre B, Hoffmann JA (1997). Antimicrobial peptide defense in Drosophila. Bioessays 19: 1019-1026.         [ Links ]

30. Menzel R (2001). Searching of the memory trace in a mini-brain, the honeybee. Learn Mem. 8: 53-62.         [ Links ]

31. Moncada S, Palmer RMJ, Higgs EA (1991). Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev. 43: 109-142.         [ Links ]

32. Müller U (1997). The nitric oxide system in insects. Prog Neurobiol. 51: 363-381.         [ Links ]

33. Muta T, Iwanaga S (1996). The role of hemolymph coagulation in innate immunity. Curr Op Immunol. 8: 41-47.         [ Links ]

34. Nappi AJ, Ottaviani E (2000). Cytoxicity and cytotoxic molecules in invertebrates. Bioessays 22: 469-480.         [ Links ]

35. Nappi AJ, Vass E (1993). Melanogenesis and the generation of cytotoxic molecules during insect cellular immune reactions. Pigment Cell Res. 6: 117-126.         [ Links ]

36. Nappi AJ, Vass E, Frey F, Carton Y (1995). Superoxide anion generation in Drosophila during melanotic encapsulation of parasites. Eur J Cell Biol. 68: 450-456.         [ Links ]

37. Nappi AJ, Vass E (1998). Hydrogen peroxide production in immune-reactive Drosophila melanogaster. J Parasitol. 84: 1150-1157.         [ Links ]

38. Nappi AJ, Vass E, Frey F, Carton Y (2000). Nitric oxide involvement in Drosophila immunity. Nitric Oxide: Biol Chem. 4: 423-30.         [ Links ]

39. Ott SR, Elphick M (2003). New techniques for whole-mount NADPH-diaphorase histochemistry demonstrated in insect ganglia. J Histochem Cytochem. 51(4): 523-532.         [ Links ]

40. Ratcliffe NA (1986). Insect cellular immunity and the recognition of foreignness. Symp Zool Soc Lond. 56: 21-43.         [ Links ]

41. Ratcliffe NA, Rowley AF (1979). Role of insect hemocytes against biological agents. In: Insect hemocytes: development, forms, functions and techniques. Gupta  AP (Ed). Cambridge Univesity Press, 31-414.         [ Links ]

42. Ratcliffe NA, Rowley AF, Fitzgerald SW, Rhodes CP (1985). Invertebrate immunity, basic concepts and recent advances.  Int Rev Cytol. 97: 183-349.         [ Links ]

43. Regulski M, Tully T (1995). Molecular and biochemical characterization of dNOS: A Drosophila Ca²⁺/calmodulin-dependent nitric oxide synthase. Proc Natl Acad Sci. USA. 92: 90722-90726.         [ Links ]

44. Ribeiro JMC, Nussenzveig RH (1993). Nitric oxide synthase activity from a hematophagous insect salivary gland. FEBS 330: 165-168.         [ Links ]

45. Rimele TJ, Armstrong SJ, Grimes D, Sturm RJ (1991). Rat peritoneal neutrophils selectively relax vascular smooth muscle. J Pharmacol Exp Ther. 258: 963-971.         [ Links ]

46. Rizki TM, Rizki RM (1987). Surface changes on hemocytes during encapsulation in Drosophila melanogaster. In: Hemocytic and humoral immunity in arthropods. Gupta AP (Ed). New York: Wiley Interscience, pp. 157-190.         [ Links ]

47. Salter M, Knowles RG, Moncada S (1991). Widespread tissue distribution, species distribution and changes in activity of Ca²⁺dependent and Ca²⁺-independent nitric oxide synthases. FEBS Lett. 291: 145-149.         [ Links ]

48. Slovák M, Kazimírová M, Bázliková M (1991). Haemocytes of Mamestra brassicae (L.) (Lepidoptera, Noctuidae) and their phagocytic activity. Acta Entomol Bohemoslov. 88: 161-172.         [ Links ]

49. Springall DR, Riveros-Moreno V, Buttery L, Suburo A, Bishop AE, Merrett M, Moncada S, Polak JM (1992). Immunological detection o nitric oxide synthase(s) in human tissues using heterologous antibodies suggesting different isoforms. Histochemistry 98: 259-266.         [ Links ]

50. Stefano GB, Ottaviani E (2002). The biochemical substrate of nitric oxide signaling is present in primitive non-cognitive organisms. Brain Res. 924: 82-89.         [ Links ]

51. Wells JD (1991). Chrysomya megacephala (Diptera: Calliphoridae) has reached the continental United States: review of its biology, pest status, and spread around the world. J Med Entomol. 28:  471-473.         [ Links ]

52. Wells JD, Kurahashi H (1994).  Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) development: rate, variation and the implications for forensic entomology. Jpn J Sanit Zool. 45: 303-309.         [ Links ]

53. Whitten MMA, Mello CB, Gomes SAO, Nigam Y, Azambuja ES, Garcia ES, Ratcliffe NA (2001). Role of superoxide and rective nitrogen intermediates in  Rhodnius prolixus (Reduviidae)/ Trypanosoma rangeli interactions. Exp Parasitol. 98: 44-57.         [ Links ]

54. Wiesner A (1991). Introduction of immunity by latex beads and by hemolymph transfer in  Galleria mellonella. Dev Comp Immunol. 15: 241-250.         [ Links ]

55. Wiesner A (1992). Characteristics of inert beads provoking humoral immune responses in Galleria mellonella. J Insect Physiol. 38: 533-541.         [ Links ]

56. Wildemann B, Bicker G (1999). Developmental expression of nitric oxide/cyclic GMP synthesizing cells in the nervous system of Drosophila melanogaster. J Neurobiol. 381: 1-15.         [ Links ]

57. Yuda M, Hirai M, Hirai K, Matsumura H, Ando K, ChinzeiY (1996). cDNA cloning, expression and characterization of nitric oxide synthase from the salivary glands of the blood-sucking insect Rhodnius prolixus. Eur J Biochem. 242: 807-812.         [ Links ]

Received on March 20, 2006. Accepted on February 12, 2007.