Beating the mosquito at its own game

When tackling one of the world’s most prolific killers – a bit of mimicry was just the thing to lure this airborne disease spreader

Aedes aegypti – the most prolific vector of the arboviruses causing dengue and yellow fever

BIRD flu and other zoonotic diseases transmitted directly from animals to man are now paraded as the new big ‘killers of the future’, but insect-vectored diseases are still the biggest ‘killers of today’. And none more so than human parasites and viruses causing malaria, dengue fever, yellow fever and West Nile virus (WNV) borne, carried and transmitted by Anopheles, Aedes and Culex mosquitoes.
   Success of mosquitoes as vectors of human disease hinges on their ability to preferentially seek out, pin-point and target human (over animal) hosts. This is achieved by strong attraction to specific natural profiles of volatile chemicals excreted in sweat on to the skin surface of the human body.
   AgriSense-BCS (United Kingdom) and BioGents GmbH (Germany) are two companies dedicated to technologies for insect pest monitoring and control, using highly specific chemical attractants and insect traps custom-designed to intrinsic behavioural patterns of target insects. They have jointly developed a commercial lure/dispenser for deployment in a novel and unique insect trap. The system is already providing an accurate and efficient monitoring of mosquito populations by playing and beating these blood-sucking insects at their own game.
   The lure is the result of years of research at the University of Regensburg, Germany and is based on a specific blend of chemicals identified in human sweat and therefore highly specific for anthropophylic (human biting) insects. The attractant blend is contained in commercial lure/dispenser designed and produced by AgriSense for use in a commercial trap called the BG-Sentinel, developed and produced by BioGents to mimic convection of volatile chemicals from a sweat-laden human body.
   Both companies are research-based with many decades of experience in the design, development and deployment of insect attractants (lures) and custom-designed traps for insect pest monitoring and control in a wide range of fields including public health.

BG Sentinel Trap.  – The yellow arrows show the direction of the drawn-in air, the red arrows the air loaded with the attractants which are emitted through the permeable fabric.

Most field research and development work has been carried out with Aedes, a medically-important genus belonging to the Culcini. The group includes some of the most mobile, versatile and potentially lethal species of mosquito found anywhere in the world, carrying and spreading the most notorious lethal arboviruses known to man. Assisted by international trade and travel and nurtured by global warming Aedes mosquitoes are moving into and surviving in hitherto uncongenial and, therefore, unexploited regions of the world.
   There are many different species of Aedes displaying effects and consequences that range from simple nuisance value against tourists to vectors of life-threatening disease. Of the hundreds of different species there is one (Aedes aegypti) with a traditionally tropical distribution and which stands head and shoulders above the rest as a vector of human disease.
    Aedes aegypti is the most prolific vector for arboviruses causing yellow fever, and dengue fever considered to be the most health (life) threatening and economically important arbovirus in the world, and responsible for an increasingly large number of deaths particularly in Asia and South America.
   In Asia and the Americas, Aedes aegypti breeds primarily in man-made containers like earthenware jars, metal drums and concrete cisterns used for domestic water storage, as well as discarded plastic food containers, used automobile tyres and other items that collect rainwater. Such is the seriousness of Dengue carrying mosquitoes in Brazil that use of water containers to support fresh flowers in cemeteries and grave yards is banned.
  Aedes mosquitoes are highly adaptable insect breeders traditionally selecting clean and fresh or only slightly polluted water, including puddles left after rainfall and in which eggs can survive when the puddle dries up. The female Aedes mosquito is a day-time feeder. This feeding behaviour heightens and broadens health risks compared with night-time feeders like Culex pipiens, the so called ‘house’ mosquito which has been primarily responsible for spread of West Nile Virus (WNV) across the United States of America. Day-time feeding behaviour of Aedes makes this mosquito an ideal subject for the research and development into new insect attractants and trapping systems for pest monitoring and control.

A range of methods have traditionally been used to assess mosquito numbers and  accompanying health hazards and risks, though few are sufficiently and universally sensitive for the degree of accuracy required.
   Assessments are made using counts and records of larvae and pupae at breeding sites, or adults by searching homes and dwellings and vacuuming up all mosquitoes using special-back pack aspirators. This may prove accurate for species that stay in the dwelling after taking a blood meal, but the system will also ‘suck up’ nuisance species of no medical importance.
  Custom-designed traps that lure and encourage gravid females to lay eggs on or inside the trap surface is a major step forward, but there is no way of knowing which mosquito species has laid the eggs without laboriously hatching and identifying the larvae.
   The only way to accurately assess the real potential for disease transmission both temporally (in time) and spatially (in place) is to target adult female mosquitoes as they seek out human hosts for a blood meal. 
  

BG Sentinel trap in position

The air-borne adult female mosquito seeking a blood meal is a ravenous beast with its single-minded purpose achieved through attraction to the desired target hosts. Mosquitoes take blood meals from the widest range of animals, including mammals and birds, but humans are invariably the preferred host.
    Human volunteers are used as living, breathing, sweating landing and collection sites for host seeking female mosquitoes. In doing so they are literally sacrificed to blood sucking mosquitoes and perhaps disease, through spending several hours in a test area and catching all female mosquitoes that land on their skin. Apart from being highly questionable the method cannot be properly standardised because different individuals have different capacities to attract mosquitoes.
    The unethical and inaccurate aspects of using the human body for this system are overtaken by development of attractants and lures based on actual chemical components of human perspiration, and deployed in a trap that mimics release of volatile sweat chemicals from the human body.

Twin centre-pieces of this novel and commercially operating system are the lure/dispenser which contains a crafted blend of human sweat components for anthropophylic insects, and the unique and novel custom-designed trap. The mixture of attractant was researched at the University of Regensburg in Germany and the controlled release technology was developed by AgriSense in the United Kingdom.
   Specific chemicals given off by human sweat deposited on human skin surfaces were identified and subsequently assessed for mosquito attractant properties and potential. Researchers used the ‘Y’ tube bioassay system (choice olfactometer) to determine the role and effect of these chemical factors in chemical and sensory ecology of the mosquito species under test. Through sequential activation, upwind flight and odour choice, the research team were able to first identify the chemicals and then copy and craft the most potent attractant blend for use in a commercial lure.
   Emanations from vertebrate hosts including humans comprise complex blends of chemicals. Odour blends rather than individual constituent chemicals were shown to attract A.aegypti. Highly attractive blends consisted of L-(+)-lactic acid, ammonia and specific fatty acids which were essential to maximise attractant properties of the blend. In a direct competition bioassay a distinct blend was shown to be as attractive as an average human hand.
   Some human odour samples were less attractive than others but addition of lactic acid significantly increased their degree of attractiveness. While there was almost no response by A. aegypti to natural animal odour samples addition of lactic acid made these animal samples as attractive as natural human odour samples. Lactic acid as a component of the blend is the single most important factor in attraction of mosquitoes to human odour samples. Carbon dioxide did not prove essential to effectiveness of the blend but enhances catching rate through a synergistic effect by instant sensitisation of A. aegypti to human skin odours. Water also displays synergistic effects.
   The commercial lure produced by AgriSense is custom-designed (physically and chemically) as the attractant dispenser for a new suction trap designed, commercialised and patented by BioGents.
  The appropriately named ‘BG Sentinel’ trap mimics convection currents created and generated during heat exchange by the human body, as well as displaying attractive visual (black and white) clues, and releasing attractant chemicals from the lure over an extensive surface area.
  An integral fan blows volatile chemicals upwards through the fabric gauze on top of the trap in a convection-like current, thus mimicking scents continually emitted by sweat on the skin surface of the human body. Any mosquitoes following the scent trail are visually attracted to the edge of the black suction column centrally placed in the top of the trap and surrounded by the white fabric gauze. Insects are subsequently forced into a net inside the trap by the downward suction forces generated by the trap’s fan.

Aedes turned out to be an easy ‘lab animal’ to work with. Day-flying eliminates the need to use the red light techniques required to observe and monitor activity of night-flying species like Culex and Anopheles. But this is not the reason why these researchers selected Aedes as prime target for monitoring and control of arthropod vectors of human disease.
   Dengue in tandem with the Dengue Haemorragic Fever (DHF) complication is a killer disease, currently on the rampage across huge swathes of tropical and sub tropical Asia, Africa and America. The primary vector of Dengue is Aedes aegypti a predominantly urban species exploiting huge numbers of people crowded together in Third World city slums who are at continual risk. Preference for breeding indoors and requiring a minimum of fresh water, whether from leaking taps or other sources, aggravates already high levels of disease transmission.
   Disease incidence is highest during periods of high temperature and high rainfall. Evidence suggests the vector is continuing to adapt to its preferred urban environment by now using polluted water sources for breeding if a fresh water source does not exist.
  Many other mosquitoes that leave a raft of eggs in one pool of water but female A.aegypti hedges her bets by laying her eggs at many different sites. Unlike other mosquitoes which tend to feed from just one animal or human, female A. aegypti takes blood meals from several or more different hosts and has the potential to infect up to 10 people with Dengue. These factors also contribute to the status of A.aegypti as the primary vector of Dengue and DHF.
  There are four different virus serotypes (DEN 1, DEN 2, DEN 3 and DEN 4) that cause Dengue. Recovery from one confers life long immunity to that serotype but only partial and temporary protection against the other three. What’s more there is good evidence to suggest sequential infection increases the risk of contracting the more serious disease resulting in DHF.
   Dengue is endemic in more than 100 countries spanning South East Asia and Western Pacific, Middle East and Eastern Mediterranean, Africa and the Americas, putting more than 2500 million people at risk. The WHO estimates there could be as many as 50 million cases each year. Not only is the disease spreading into new areas and putting more people at risk, but is occurring in explosive outbreaks. The 609,000 cases (of which 15,000 were DHF) reported throughout the Americas during 2001 represented a doubling of cases over 5 years.
  Dengue is sweeping South East Asia in spite of aggressive attempts to control vector and disease on course to be the worst ever outbreak and surpassing 1998 when 750,000 cases were recorded. Dengue is traditionally regarded as a disease of the poor and poverty stricken neighbourhoods but no country has escaped the 2007 outbreak. Singapore and Malaysia are fighting the disease alongside Laos and Cambodia. By August 2007 Indonesia had already recorded 100,000 cases and 1000 deaths with Jakarta predicting an eventual 200,000 cases compared with 114,000 last year. Malaysia has suffered a 50% increase in cases (56 deaths) with 1000 people infected weekly during June and July. Vietnam, which traditionally suffers high rates of dengue, had recorded 33,000 (32 deaths) representing a 40% increase on 2006.
  Singapore has one of the lowest fatality rates in the region but 3,000 plus cases translates into one of the highest infection rates on a per capita basis. Australia managed and eliminated Dengue outbreaks in previous years in the northern tropical region around Cairns, but is particularly concerned about re-introduction this year given record numbers of cases elsewhere in the Asia Pacific region.
   Dengue has been described as an enigmatic virus, difficult to diagnose and impossible to quarantine. Symptoms and antibodies take many days to appear which makes Dengue difficult to detect and treat in time. The only logical alternative is to target the mosquito which carries and transmits the virus.

AgriSense Lure – Mosquito attractant in lure for the BG Sentinel trap

The BG-Sentinel-lure combination has been tested in Brazilian cities for monitoring A. aegypti in the classic urban environments. Female Aedes mosquitoes are active during the day and virtually impossible to catch and secure with conventional trapping systems using only carbon dioxide.
   Field trials in the Brazilian city of Belo Horizonte showed the BG-Sentinel-lure system to outperform other traps (gas-powered carbon dioxide trap and visual-cue only trap) and to be as efficient as the real thing (live human landing/biting collections). A six-month comparison between BG-Sentinel and a sticky ovitrap for gravid females showed BG-Sentinel to be far more efficient and sensitive for measuring density of A. aegypti populations.
  BG-Sentinel trapping system is more efficient in sensitivity as well as absolute numbers of mosquitoes caught. Traps deployed for several months at a single location in Belo Horizonte showed catch rates rising perceptibly at the beginning of the wet season. However, the trap was sufficiently sensitive to catch enough mosquitoes during the dry season and enable assessment of these much lower seasonal populations.
   Trials are part of the on-going strategy to develop and deploy early warning systems for Dengue fever and to monitor effectiveness of any control measures. Both early warning systems and control measure monitoring systems require ultra-sensitive trapping systems that can lure and secure mosquitoes even when they are in low numbers, and therefore detect small early-stage breeding populations. 
   Another major benefit afforded by the BG-Sentinel is better use of human resources and ability to increase the intensity of pest monitoring. Brazilian city authorities have only been able to carry out mosquito egg/larval surveys at a rate of four per district in any one calendar year. But mosquitoes have a development cycle of just two weeks which means there is no way of providing a completely up to date picture of disease risk with just one survey every 12 weeks. This new technology provided by the BG-Sentinel and armed with the attractant blend will allow authorities to survey weekly with the same level of human resources.
   The system is being evaluated in the coastal city of Cairns in tropical northern Queensland, where previous Dengue outbreaks have occurred (including 1998 and 2003) although all were quickly stamped out. That said the Queensland State and national government are continually worried about re-introduction and want to develop a reliable and efficient early warning system. They require an ultra sensitive trapping system to detect small populations of Aedes mosquitoes in the very earliest stages of development. The University of Queensland and the Brisbane Public Health Department have already tested the system with exceptionally good results.
   The United States military with personnel in many Dengue infected areas of the world are testing the BG Sentinel system in a range of countries including Thailand, Indonesia and Peru.
   First field trials were to evaluate the system for mosquito monitoring only, but work is now underway in Brazil to use the system as a control tool, reducing incidence of Dengue by exploiting weak points in the A. aegypti/Dengue life cycle and transmission period. Researchers are fine-tuning the strategy to hit the disease just as much as the vector for preventing transmission of Dengue from mosquito vector to human host.
   It only takes 7 days for A. aegypti eggs to develop into mosquitoes but 12 days for an adult female mosquito (having acquired infected blood) to transmit the disease to a new host. This is considered a long time for adult Aedes mosquitoes to remain alive.
   Population models show control authorities will not have to trap that many mosquitoes to exert a significant effect on disease transmission. The research group in Germany has received funding to test the theory in Brazil where they are going into villages and deploying as many traps as possible to see if they can break the transmission cycle. Early calculations show that removing just 35% of the Aedes mosquito population should reduce level of Dengue by 80%. Two key factors in the calculations are the relatively long period (12 days) required before the mosquito becomes infective, after sucking up infected blood, and corresponding short life expectancy of the same adult mosquitoes.
   Plans are underway to extend application of the BG-Sentinel-lure system to monitoring and control of other species of Aedes mosquito responsible for carrying different diseases, as well as Aedes carriers of Dengue into new hitherto disease-free temperate areas of the world.
   Aedes albopictus (Asian tiger mosquito), a secondary vector of Dengue has spread alarmingly to establish in the United States, several Latin American and Caribbean countries and parts of Europe including Italy. Rapid geographic spread of this species is largely attributed to international trade in used tyres. Unlike A. aegypti, A. albopictus breeds mostly outdoors in gardens and other leafy areas which is one reason why it is secondary to its close relative as a disease vector.
   Nevertheless A. albopictus is considered a real threat and capable of bringing Dengue, previously considered a purely tropical disease, in to the temperate areas of the North America and Europe. These new threats are exacerbated by global warming, allowing both A. albopictus and A. aegypti to extend their normal traditional range.
   The BG-Sentinel-lure is the only system that effectively traps and monitors A. polynesiensis (Polynesian tiger mosquito), carrying and transmitting the parasitic roundworm (nematode) causing filariasis in the Asia-Pacific region. A. polynesiensis is an important vector of Dengue in this particular region.
   The US military are evaluating the system for deployment in Iraq and Afghanistan against sand flies (phlebotominae), which carry and transmit leishmaniasis. Researchers are encouraged by the good results already achieved in Mali and Ghana against sand flies. BG Sentinel and loaded with the new attractant blend has also been evaluated for monitoring Culex pipiens, the primary vector of WNV in North America.
  
By Dr Owen Jones. Dr Jones s the General Manager of AgriSense BCS Ltd, a company which develops insect monitoring and control systems.