Blood-feeding, or hematophagous, arthropods transmit numerous types of parasites and infectious agents that increase human mortality worldwide. Blood sucking is risky for these insects but provides them with rich protein meals. Hematophagous arthropods feed on blood by either piercing blood vessels or by causing blood to pool. Vessel feeders have fine, piercing stylets that probe the skin until a small capillary is found. Pool feeders instead have mouth parts adapted for cutting the skin and creating an extra vascular puddle of blood which here to feed from that forms intradermally, or on the host’s surface.

Vector saliva contains substances that offer potential pharmacological uses for humans. These compounds include anti-hemostatic, vasodilatory, anticoagulant, anti-inflammatory and immunomodulatory substances. The presence of such diverse compounds in vector saliva stimulates researchers to search for novel and potent pharmaceuticals and vaccines.

Many of these same compounds help blood-sucking insects evade infection by the parasites they are transporting. Salivary gland lysates of the sand fly, Lu. Longipalpis, for example, inhibit several macrophage functions in the fly. Thus, the fly's immune system cannot detect a parasite that the fly is transmitting. The tick, human louse and sandfly contain potent immunomodulators in their saliva. These immunomodulators counter their host’s haemostatic, inflammatory and immune responses to facilitate blood feeding. Immunomodulators prevent the host from rejecting the insect that is blood feeding, and they enhance pathogen transmission by insects. The louse stores immunosuppressive properties that can prevent non-desirable microorganisms from invading an open wound on skin. Bioactive salivary factors exist in the salivary glands of Culicoides variipennis (the biting midge). The salivary antihemostatic and immunomodulatory activities facilitate blood feeding but also enhance the infectivity of the diseases Bluetongue and Vesicular stomatitis.

Hematophagous insects feed from a wide range of different host animals. For obvious and painful reasons, humans know that many of them feed from mammals, but many other host animals are also exploited, including birds, reptiles, amphibians, fish, and even insects, arachnids and annelids (Hocking 1971).

Researchers now know that the flying bloodsuckers are not simply flying syringes and that administering pathogens to animals by simple injection does not mimic natural disease transmission. These blood feeders produce bioactive molecules in their salivary glands that are inoculated into host skin via saliva during feeding. The salivary bioactive molecules have antihemostatic and immunodulatory functions (Ribeiro 1995). Such functions allow hematophaghous insects to live with and contend with the host immune system. Vector salivary secretions have been shown to modulate host immune responses (Theodos and Titus 1993), and arthropod-borne pathogens may benefit from the effects of vector salivary factors that are primarily involved in blood feeding (Champagne 1994). These effects may influence the pathogenesis and epidemiology of arthropod-borne diseases (Warburg et al 1994).

Because saliva is the only thing an arthropod injects during the blood-feeding process, salivary proteins must be responsible for insects' ability to defeat mammals' blood coagulation response. Paesen et al. (2000) reviewed an example of a blood-feeding insect's ability to do this. They studied the tick histamine-binding proteins in tick saliva. Histamine has an important role in inflammation and allergy. Histamine can lower blood pressure, dilate capillaries and increase blood flow.

Another example of an insect's ability to prevent coagulation exists in the saliva of mosquitoes, which have vasodilators in their saliva. Pappas et al. (1986) first noted the salivary vasodilator of Aedes aegypti. Later, Ribeiro (1992) discovered that these vasodilators were tachykinins. Tachykinins are peptides that have diverse pharmacological actions in the central nervous system, cardiovascular, and glandular tissues.

Many ectoparasitic arthropods have compounds that help them evade the effects of host grooming, a process that represents a significant threat to ectoparasitic arthropods, especially for long-term blood feeders. For example, salivary glands of the tick, Ixodes dammini, contain a kininase. This enzyme is capable of destroying bradykinin, a mediator involved in the sensation of the itch (Ribeiro et al. 1985). Decreasing host awareness of the insect on the skin surface increases successful parasitism.

In the second possible route, insects that had no preadaptions for blood feeding may have gradually developed a more intimate association with potential vertebrate hosts. They would have done this by first feeding on the organic matter in nests, then moving on to dung and dead skin particles from the host, and finally consuming live skin and blood.

In the case of either route, blood feeding required morphological adaptations. These included developing morphological adaptations, such as developing mouthparts able to penetrate skin; physiological adaptations, such as the developing proteolytic enzymes for blood digestion; and behavioral adaptations, such as developing the ability to find objects that have blood and distinguish them from objects that do not. At the same time, the mosquitoes have had to deal with unwilling blood donors (such as people) that display defensive behavior that both discourage mosquitoes from feeding and create a source of significant insect mortality (Edman and Scott 1987).

Generally, there are two types of blood feeders: vessel feeders and pool feeders. Vessel feeders take blood directly from venules or small veins, while pool feeders take blood from a blood pool resulting from the laceration of blood vessels (James and Harwood page 24-25). In most blood-feeding insect species, blooding behavior is confined to insects in the adult stage, most often females. For instance, only adult female mosquitoes take blood meals; they also take flower nectar. The male mosquitoes feed solely on nectar, and the larvae are aquatic filter feeders. In this case, the primary utilization of blood is for developing eggs. On the other hand, blood provides all the nutritional needs for insects such as the kissing bugs, in which both sexes, and nymphs as well as adults, blood feed (Edman and Eldridge, p. 57).

Anticoagulants are substances that prevent the clotting of blood. Coagulation is desirable in vertebrates when they become injured; it prevents unnecessary blood loss. Thrombin is a blood coagulation enzyme often found in insect saliva. Vasodilators can make blood vessels larger, allowing for more blood flow. Vasodilatory substances have been identified in ticks; nitric oxide-binding proteins are present in triatomine bugs, and novel vasoactive peptides in exist in sand flies and mosquitoes (Champagne, 1994).

When a vector probes for blood, tissue is damaged, resulting in the activation of platelets and vasoconstriction. These hemostatic processes occur within seconds and are countered by potent salivary platelet anti-aggregating factors and vasodilators. Stark and James (1996) used in vitro clotting assays with plasma to examine eight mosquito species for the presence of anticoagulants in salivary gland extracts. Independent parts of the coagulation cascade were assayed in the presence of salivary gland extracts to determine where in the cascade the anticoagulants exerted their inhibitory effects. Results indicated that members of the malaria vector, Anophelinae, produced thrombin-directed anticoagulants, while members of the Culicinae, possessed Fxa-directed anticoagulants. This finding suggests that: 1) hematophagy comes before the separation of major mosquito subfamilies and the different activities reflect adaptations to preferred hosts, or 2) blood feeding evolved independently after the separation of the subfamilies.

Researchers have also studied the potency of anticoagulants in mosquitoes. For instance, the filariasis vector, Culex quinquefasciatus, exhibited an anticoagulant activity twice as high as that of Aedes aegypti. New World Culex are commonly thought of as bird feeders that have expanded there host range to include humans and other vertebrates. The observed differences between the two mosquito species may reflect the fact that bird hemostasis relies more heavily on coagulation than does mammalian hemostasis (Campbell and Dein, 1984).

A soft tick called Ornithodorous moubata was studied by (Hellmann an Hawkins 1967) for anticoagulants. Initially, this tick was shown to have a heat-labile, thrombin-directed anticoagulant activity in the salivary glands, gut, coxal fluid and egg extracts; this activity was present in all developmental stages (Hellmann and Hawkings 1967). When the tick extracts were incubated for 40 degree Celcius for 15 minutes, the salivary glands still exhibited an anticoagulant activity that prolonged the aPTT, but not the PT, clotting assays. Incubation inhibited FIXa by reconstituting FIX-deficient plasma that had been pre-incubated with the salivary gland extract. Physical and chemical profile analyses showed that the two anticoagulants were different molecules. An additional study on this tick was done by Waxman et al. (1990). They isolated Fxa-directed anticoagulant and characterized from whole bodies of O. moubata an anticoagulant called TAP (tick anticoagulant peptide).

Horn fly saliva also targets thrombin or cooagulation action in hemostasis. The horn fly, Hematobia irritans, is a troublesome livestock pest because the adult stages of both sexes are aggressive blood feeders. Remarkably, even though horn fly adults feed recurrently on their host as ectoparasites, these flies lack the ADP-responsive antiplatelet aggregation and vasodilator antihemostatic systems described for other blood-feeding Diptera. Horn fly salivary gland extracts do interfere with the normal coagulation process, however, as demonstrated by the recalcification time assay. (Cupp et al., 2000). Thus, as compared with other zoophilic Diptera such as black flies, the horn fly inhibits coagulation of blood.

Biting midges (Culicoides variipennis) have evolved salivary components that counteract hemostatsis. The host forms a platelet plug forms within seconds after the wall of small blood vessels is disrupted. Adenosine diphosphate (ADP), however, is a potent agonist of platelet aggregation. An apyrase in biting midge salivary glands degrades ADP to AMP and inorganic phosphate (Leon &Tabachnick, 1996). This increased blood flow resulting from the action of the salivary vasodilator could facilitate blood feeding.

A salivary nitrophorin (nitric-oxide-carrying hemoprotein) helps two blood feeders to feed on their vertebrate hosts. Nitrophorins are haemoproteins found in saliva of bed bugs (Cimex lecturlarius) and conenose bugs (Rhodnius prolixus). Valenzuela and Ribeiro (1998) found that salivary nitric oxide promotes vasodilatation and inhibits platelet aggregation.

The human body louse (Pediculus humanus) also has anticoagulants, but the compound and glandular source have not been determined. Mumcuoglu et al. (1996) characterized an anticoagulant activity in thoracic extracts containing two kinds of glands, but as yet the specific glandular source of the activity has not been determined.

Immnomodulatory Activities

Immunomodulating substances alter, suppress or strengthen the body's immune system. Charlab (2000) found salivary adenosine deaminase from the sand fly, Lutzomyia longipalpis. Adenosine deaminase is an enzyme ubiquitous in mammalian tissue. People who lack this enzyme become immunosuppressed. In the process of sequencing a subtracted cDNA library from the salivary glands of the sandfly mentioned above, Charlab found cDNA with similarities to gene products of the adenosine deaminase family. Charlab points out that the possible role for this enzyme is converting adenosine to inosine during blood feeding of the sandfly. Continued studies on this enzyme and its process could lead to the discovery of useful pharmacological materials.

The black fly, Simulium vittatum, causes economic losses by reducing beef and milk production in cattle, reducing the efficiency of agricultural and industrial workers, and spreading disease. Simulium vittatum is a quick feeding insect that modulates host defenses. The specificity of the antibody response to salivary gland antigens of this fly was investigated by comparing the responses of mice bitten by flies to the responses of mice immunized with fly salivary gland extract (Cross et al., 1993a). Mice bitten by the fly developed antibodies to fewer salivary gland molecules than did immunized mice.

This finding indicates that molecules in the salivary gland extract used for immunization were either absent from the saliva introduced with the bite or were poorly immunogenic in the context of total saliva. Antibodies produced by mice immunized with black fly salivary gland extract were of the IgM, IgG, and IgE classes, which implies that both circulating antibodies and IgE allergic reactions mediate immune response. Both fly-bitten and immunized animals developed IgE antibodies, which contribute to immediate allergic responses to fly bites.

The salivary gland of Simulium vittatum is immuno suppressive (Cross et al. 1993b). Mice inoculated with this extract have fewer spleen cells expressing class II major histocompatibility complex (MHC) surface antigens than do non-inoculated mice. These molecules are important for the presentation of immunogens to T lymphocytes by macrophages and other antigen presenting cells. Cross et al. (1993b) found that the same Simulium vittatum salivary extract inhibits the ability of B and T lymphocytes to proliferate in vitro in the presence of mitogens. Mitogens are substances, which cause cells, particularly lymphocytes to undergo cell division.

Additionally, immunization of mice with sheep red blood cells combined with black fly salivary gland extract resulted in a more intense antibody response than immunization with sheep red blood cells alone (Cross et al. 1993b). This suggests that black fly saliva contain factors than can enhance certain immune functions. To investigate how Simulium vittatum might affect the immune response, mice were inoculated repeatedly with salivary gland extract or saline before receiving an injection of ovalbumin (Cross et al. 1994). Ovalbumin is an antigen that elicits an immune response in animals with functional T lymphocytes. Results indicated that lymphocytes derived from mice inoculated with the fly salivary extract before injection with ovalbumin produced less of the cytokines interleukin –5 (IL5) and interleukin-10 (IL-10) in vitro than did lymphocytes from saline pre-inoculated controls (Cross et al. 1994). Preinoculation of mice lymphocytes with salivary gland extract did not affect the ovabumin-elicited production of three other cytokines: interleukin-2 (IL-2), interleukin-4 IL-4) and interferon-gamma. All these findings indicate that this quick-feeding fly of medical importance can down-regulate host defenses.

Evidence indicates that saliva protects the parasites and thus augments infectivity of the host. For instance, to induce infection with Leishmania major in experimental mice, investigators commonly inject millions of parasites with a syringe. The sand fly, in contrast, creates a Leishmania major infection by injecting only about 1-100 parasites (Warburg and Schlein 1986). Researchers wanted to know why the sandly is more efficient at creating an infection compared to the mechanical syringe technique. They also wanted to learn why the low number of parasites survives the hemostatic processes of the host.

Because the sand fly injects the parasite while injecting saliva into the host, it is possible that something in the saliva helps the parasite survive the host defenses. To test this possibility, Titus and Ribeiro (1988) tested mice infected with Leishmania major in the presence or absence of sandfly salivary gland lysate to determine whether the presence of saliva enhances the infectivity of Leishmania major for mice. Lysates are phage particles released from the host and help fight off non-desirable foreign particles. Results showed that in addition to enhancing lesion size, sandfly salivary gland lysate markedly enhanced the parasite burden within the lesions. Sometimes the parasite burden in saliva-treated lesions was 5580 times greater than the parasite burden found in the control or saliva-free lesions.

Arthropod modulation of host immunity likely evolved to facilitate blood feeding or tissue parasitism. Natural cycles of arboviral diseases between arthropods and mammals depend on the transmission of arboviruses to mammalian hosts during the act of vector blood feeding. Blood feeding is risky for insects because vertebrates are very large; attempting to blood feed could be lethal. However, the nutritional materials derived from blood can support present and future life for the insect.

Bioactive salivary compounds exist in the salivary glands of ticks, mosquitoes, sand flies and many other vectors. These compounds have anticoagulant, antihemostatic and immunomodulatory properties that facilitate blood feeding. Blood-feeding insects can suppress host innate and specific acquired immune defenses. Thus, insect blood feeding should be thought of in a continuum over time instead of as an individual and sometimes painful event. The fact that insect saliva can enhance pathogen transmission leads to the possibility that vaccinating the host against the components of vector saliva will inhibit pathogen transmission. Research is now taking into account these critical interactions between insect saliva and blood feeding to develop a more realistic representation of arthropod-borne diseases. This knowledge could lead to useful tools to develop novel control strategies aimed at reducing the impact of arthropod-borne diseases worldwide. An additional benefit of research could come from the medical community's exploration of the use of insect salivary compounds for potential pharmalogical uses.