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Abstract: With an increased incidence of arbovirus transmission, and an ineffective pest management approach, there has been no more important time to understand the physiological and chemical cues used for attraction by disease vectors.  The mosquitoes  of the Anopheles and Aedes genii present a good model for study due to their abundance, behavior, and impact on human health.  Experiments to determine long-range orientation, selection of biting sites on a host and chemical and visual cues associated with attraction are all hot topics to date.  Recent discovery has led to the elucidation of many simple, and many complex chemicals used by mosquitoes for orientation.  Along with a broad base of chemicals for mosquitoes to orient toward, odor-proteins have also been determined to aid mosquitoes in biting site selection.  However, sensory physiology of mosquitoes also include the ability to detect small variations in temperature.  This, in combination with odor-protein detection gives the mosquito a small region with which to preferentially seek a blood meal.  Subsequent modification of the host profile results in significant changes of these preferences.  In effect, studies of all chemical constituents that have any effect on host-seeking behavior would be of value.  The same is true for examination of auditory and visual cues as well.   Investigation of vector-parasite biology is also an important area where findings about the enhanced blood-feeding behavior of such infected vectors exist.  Increased blood-feeding behaviors are seen in Anopheles punctulatus when malarial parasites are present.  The enhanced blood feeding behavior is characterized by multiple probes by the mosquito, and multiple bites if a full blood-meal is not obtained.  This finding ultimately means that host-seeking behavior might also be more highly induced when parasites are present.  However, endogenous regulation of host-seeking behavior may prove to be the most important factor in regulating spread of disease.  Although it may seem that female mosquitoes are a relentless pursuer, there are indeed times when host-seeking behaviors do cease.

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Introduction:

 Effective mosquito control is often the only means by which diseases transmitted by these insects are prevented.  Primary tactics employed against mosquito populations are the use of chemical larvacides and adulticides.  Although effective when deployed, chemicals used alone in the control of mosquitoes eventually lead to resistance and general pollution of the environment.  Often, non-target insects feel the brunt of chemical application in the form of water, soil, and plant toxicity.  Some pest-management approaches have utilized the combination of chemical or biological insecticides and natural predators of mosquitoes (J.F, Neri-Barbosa et al. 1997).  Use of Bacillus thuringiensis var. israelensis and Tolypocladium cylindrosproum (Deuteromycotina : Hyphomycetes) as an effective larvicide is currently being investigated more thoroughly (M.P. Nadeau et al. 1994).  Also, common predators of insect larvae, the Notonectidae and the Mosquito-fish, Gambusia affinis, are proving to be valuable components.

 However, transmission of arboviruses such as Dengue and Yellow Fever are on the rise in many areas of the world.  Cuba is one of the hardest hit of the regions experiencing a large resurgence of Dengue, and Yellow fever and Malaria treatments are becoming more and more ineffective.  This in concert with a growing public demand on the importance of research and testing of safer, more environmentally friendly insecticides has limited the number of approaches that can be taken.  New EPA standards have forced many current chemicals off of the market pending further research.  However, the high cost of re-registration of these chemicals is forcing the companies who produce them to search for other avenues.  Increased cost of production and the aforementioned resistance is leading the path into alternate mosquito control strategies.  None of which is more important than understanding the physiological, chemical and physical basis for the attraction of mosquitoes to their host.
 
Discussion:

Long Range Orientation:

 Breeding and egg laying sites of mosquitoes are often in remote sites, and selection of a host may be limited to species that are found close by.  However, a good degree of long range orientation is seen in hematophagous mosquitoes.  Facultatively autogenous mosquitoes of various groups are preferentially attracted to plant rather than vertebrate-host-related volatiles as newly-emerged females.  Autogenous females preference for host volatiles develops around eight to ten days.  Developmental changes in sensory responsiveness change the behavior of the female shortly after (Bowen et al. 1995).  The inherent danger in approaching a host is the primary reason for this phenomenon.  Many groups of mosquitoes postpone their blood-meal seeking behavior due to the lack of a gravid or parous condition.  This ensures that an untimely death by swatting does not occur, and ensures that the female will have time to mate and potentially feed on the blood that is necessary for egg development.  During this time, mosquitoes will actively search for nutrition in the form of plant sugars.  Under controlled conditions, mosquito females will feed on sugar preferentially for the better part of their early post-emergence development.  Sucrose and honey seem to be the main preference, but other forms of sap and sugar will suffice.

 With the newly expressed responsiveness to human and other host volatiles the host-seeking behavior begins.  Sensitivity to a variety of chemicals exists, but none is more important in long-distance orientation than carbon dioxide.  Costantini et al. show that Carbon dioxide is the single most important cue used by mosquitoes and other hematophagous insects for locating a source of blood.  Reeves (1953) was among the first to show the attracting properties of carbon dioxide and how this chemical was dose responsive in it’s action.  Since then, many approaches have been taken to increase trapping success using the naturally produced chemical.

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  Table 1.  Total response of Aedes bahamensis females to nutritional   resources (combined responses to fruit and vertebrate host) in a   two-choice olfactometer.
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 Gonotrophic Condition Age(d)  % Responding n
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 Autogenous (nulliparous)   1-2            50.0           138

 Autogenous (gravid)    8-10            52.5           120

 Parous      8-10            36.4            55

 Anautogenous                 1            27.9           197

       8-10            34.1           176
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   Table 2.  Responses of Aedes bahamensis females to a fruit-host choice   in a two-choice olfactometer.
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  Age (d)     Choice Autogenous  n     Parous     n     Anautogenous     n
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     1-2         Host      31.9 69      -------                     36.4  55
         Fruit      68.1           -------      63.6
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     8-10        Host      66.7 63       67.3 49     53.3  60
         Fruit      33.3            32.7      46.7
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 Carbon dioxide as the only attractant is still a matter of debate.  Studies have also help to clarify the role of lactic acid, ammonia, phenol, temperature and humidity (E.E. Davis et. al 1994)  Under laboratory conditions, carbon dioxide in combination with octenol (1-octen-3-ol) has been shown to increase responsiveness in many species of mosquito.  Octenol is an organic chemical produced by most vertebrates.  Some vertebrates such as oxen produce an unusual amount of the substance, and are therefore extremely enticing to a host-seeking  mosquito.  Lactic acid may not be as important in long-distance orientation, but it’s role in close-range host-seeking is believed to be extremely important.  Sensilla basiconica or grooved pegs on the antennae of the mosquito are the primary component used to sense lactic acid and carbon dioxide.  While the shorter pegs are sensitive to the lactic acid, the longer pegs are used to detect minute amounts of carbon dioxide (Bowen M.F. 1995).  Long and short pegs in the Aedes epactius are used to detect butyric acid present in the air.  Along with it’s attractive properties with respect to mosquitoes, tsetse flies are also presently being controlled using the combination of these chemicals.

Selection of Biting Sites:

 Once a host is located, the mosquito will spend little time locating the region of the host which will provide the blood-meal.  Anophelean and Aedean spp. are predisposed to areas of the host body that have an increased temperature and sweat gland density.  On a human host this results in a more frequent bite to the head and neck region along with increased biting to the legs and feet.  Although the different species differ in host preference, they all showed a significant preference to bite the legs and feet, suggesting that a mutual not specifically human factor was involved in biting site selection (Dekker T. et al.).  Dekker T. et al. also showed that the frequency of biting on the head and neck were due to increased exhalation and heat.  The same report demonstrated that the increased biting of a mosquito on the legs and feet of the host are because of the mosquito’s propensity to search for a blood-meal closer to the ground.  Subjects sitting in a stool incurred many more bites on the feet and legs, while subjects laying on the ground demonstrated a more even distribution of bites along the body.  When test subjects were raised off of the ground by a few feet, the frequency of biting decreased.  However, most species of mosquito are not fooled by the height of the subject.  Convection current and increased heat production led the mosquito to bite in an area some distance off of the ground.   B G J. Knols et al. showed that limburger cheese was an attractant to Anopheles gambiae s.s.  The odor-protein given off by this particular type of cheese was analyzed and found to be structurally similar to that of human sweat.   Knols also found that subsequent modification of host odor by removing exhaled breath and washing feet results in significant change to these preferences.
 
Visual Cues Associated with Host-Seeking Behavior:

 Mosquito vision is known to play a role in location of resources such as mates, hosts, resting and oviposition sites (S. A. Allen 1994).  Factors affecting mosquito vision include the amount of light absorbed by photopigments and time of day.  Perception of two objects side by side is based on the ability of adjacent ommatidia.  Images produced through the adjacent ommatidia are limited by the interommatidial angles of the compound eye.  Flies that are highly visually oriented generally tend to have a smaller IOA, while those that rely less on visual stimuli have a greater IOA.  Mosquitoes of the Aedes and Anopheles spp. fall in the middle of this group.  For instance, the house fly, Musca Domestica, has an interommatidial angle of ~2.4-2.8 degrees.  This compared with the 6.8 degree IOA of the Aedes group is much better at close resolution of two objects (Muir et al. 1992).  In addition, contrast sensitivity is defined as the ability of the eye to distinguish different light intensities (Mazokhin-Porshnyakov 1969).  A high value of contrast intensity signifies a higher ability to distinguish smaller differences in intensity (Snyder et al 1979).  The role of vision in long distance orientation is based mainly on ambient light in the day or dusk sky.  Studies showed for both long-range orientation (+) and short range orientation, mosquitoes discern movement, colors, shapes and patterns, with edges being particularly noticeable.   Differentiation of color and light intensity helps mosquitoes in finding sites that are conducive to host interaction.  Moreover, mosquitoes that actively seek hosts for blood meals will be attracted by artificial light sources emanating from a given point.  This in essence only serves the mosquito in general location of light sources.

Endogenous Regulation of Host-Seeking Behavior:
 
Host-seeking behavior is not always the primary goal of an adult female mosquito.  Certain brief periods in the life and daily cycle including post-emergence, post blood-meal and a nulliparous state will inhibit the behavior.  Nutritional preferences of newly emerged female mosquitoes are quite different from that of a gravid or parous female (Klowden M. 1994).  Physiological responses from the midgut are responsible for the inhibition of host-seeking in a post blood meal state.  Distention of the midgut sends signals to the brain of the insect that it can not contain any more blood.  Partial blood meals may not send the same signal to the brain, and consequently the behavior will continue.  A neuropeptide in the brain is responsible for the inhibition of the behavior until oviposition has occurred (Brown et al. 1994).  These physiological inhibitors of host-seeking behavior are compounded by a Male Accessory Gland substance that is transferred to the female during copulation.  The effect of this substance inhibits the behavior, but only in gravid females.  If a female is not in the early stages of the gonotrophic cycle, the MAG will have little if any effect. However, this behavior is not only inhibited.  The presence of malarial parasites and dengue virus particles will induce the behavior by physically blocking the entrance of blood into the gut (Koella J. C. 1996, Platt K. D et al. 1997).  Because biting rate and time spent probing the host is of importance to the spread of the disease, it would seem logical that the parasite would induce a higher biting rate and longer probe time in vectors it inhabits.  This is true for the malarial parasite Plasmodium falciparum (Koella et al. 1996) and the Dengue 2 virus (Platt et al. 1997).  The Dengue virus severely infects the salivary gland of the vector, causing a wide range of physiological response in the Aedean group of mosquitoes.

Conclusion:

 The impact of mosquitoes and their transmittable diseases plays an important role in human health.  The vectors of some of the most prevalent and costly infection are the mosquitoes of the Anophelean and Aedean groups.  Further research and more in depth looks at physiology, cytochemistry, and behavioral aspects of these world-wide pests is underway.  With the help of nations that are hardest hit by these and other dipteran insects, hopefully we can reduce the amount of fatality associated with some of the most beautiful and intriguing flies in the world.

 
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