CONTROL MEASUR2ES Vector control

1. CONTROL MEASUR2ES Vector control Medical Parasitology CBIO4500 April 24, 2012 Silvia N J Moreno

2. PARASITE CONTROL Prevention of Environmental Contamination:Antiparasite drugs. Treated hosts should be protected from re-infection. Sanitation. Destruction of Free-Living Stages:Protozoan cysts, helminth eggs and infective larvae are often extremely resistant to toxic chemicals. Destruction of Intermediate Hosts and Vectors:chemicals to kill snails, insecticides. Altering the environmental conditions so the the target species do not find the suitable habitat for its survival. Does not rely on public cooperation. Destruction of Reservoir Hosts:dogs in leishmaniasis, game animals in trypanosomiasis Prevention of Infection:Many infective stages gain entry in drinking water, Cryptosporidium, Giardia, Dracunculus etc. These can be controlled by safe water supplies. Meat inspection to prevent Taenia. Bednets prevents mosquito bites. Shoes stops hookworm larvae burrowing through the skin. Prevention of Parasite Maturation:chemoprophylaxis and vaccination Integrated Control 400,000 long-lasting insecticide-treated bed nets start the last leg of their journey from a Red Cross warehouse in Mozambique into the hands of families who need them

3. VECTOR CONTROL Major vector-borne diseases of humans, and associated aetiological agents and arthropod vectors

4. VECTOR CONTROL • Vector control has proven successful for disease control: • Malaria:eradication of malaria from most of the temperate-climate countries in the northern hemisphere • Onchocerca:the insecticide phase of the Onchocerciasis Control Prgramme (OCP) in West Africa has almost eliminated onchocerciasis from11 countries (www.who.int/ocp/index.htm) • However there is paucity of effective vector-control programmes: past neglect in this area of research, potential environmental impact of existing agents, reduction of their effectiveness because of resistance and other biological complexities of vector populations. Abandoned village near the Volta river high blindness rates resulted in the depopulation of entire villages. DTT spraying does reduce disease transmission but environmentalists have opposed to their use on large scale. Bednetsimpregnated with pyerethroidsare simple and effective at reducing vector-human contact and transmission. Limited adoption of this approach, improper use and the emergence of vector populations with resistance to pyrethroids limit their effectiveness. The result has been increased activity and productivity in many of these areas. New villages in areas formerly uninhabitable because of river blindness

5. VECTOR CONTROL Advances in vector genomics: an important advance is the sequencing of the genome of the A. gambiae mosquito (Science, 2002 298:129). The genome of Aedesaegypti (Science (2007) 316:1718-23) (yellow fever) was also sequenced, and a project is in place for the genome of Glossinamorsitans (Tsetse fly vector). This information will provide opportunities for understanding better vectors and devising new methods for their control. • Vector biology is poised to explore new strategies for vector-based disease control: • Mosquitos refractory to pathogen infection • Novel insecticides targets • Understanding the molecular basis for vector behavior, ecology and host-parasite-vector interactions

6. Manipulation of insect vector by their parasite • Parasites alter their host in some way. • Does this alteration affect transmission? • Parasite undergo a period of growth, development and sometimes reproduction within their vector: • Parasite fitness is linked to vector fitness. • Two factors that would improve parasite success: • Vector longevity • Fast Development Lutzomyia longipalpis feeding Anopheles albimanus mosquito feeding on a human arm. This mosquito is a vector of malaria and mosquito control is a very effective way of reducing the incidence of malaria.

7. Manipulation of insect vector by their parasite Host manipulation by parasite: Hematophagy:Infection results in altered vector feeding behavior. Leishmania and plasmodium. Fecundity, blood feeding, and infection: Blood is important for the vector and the parasite. Parasite-induced fecundity reduction. Allows vector survival until transmission can occur? Infection and survivorship:Do infected mosquitoes live longer?High infectivity also results in increase in mortality. Studies are not conclusive. Lutzomyia longipalpis feeding Anopheles albimanus mosquito feeding on a human arm. This mosquito is a vector of malaria and mosquito control is a very effective way of reducing the incidence of malaria.

8. Manipulation of insect vector by their parasite Hematophagy: Malaria-infected mosquitoes:Infected mosquitoes makes many attempts to feed, each time depositing parasites at the feeding site. ADP stimulates platelet recruitment to the wound site. Apyrase is an enzyme which degrades ADP and is present in the mosquito saliva and would inhibit host hemostasis promoting easier and longer blood feeding. Apyrase activity is reduced in the salivary glands of P. gallinaceum- infected Aedes Aegypti resulting in an increase in the times of the median blood location time. One study showed that P. falciparum infected An. gambiaebite more hosts and also that infected mosquitoes were most likely to take multiple meals and become fully engorged. This would increase their chances for transmission. Is infection increasing the threshold blood volume at which blood-seeking behavior is inhibited? More studies are needed in the natural infection scenario.

9. Leishmania manipulates sandfly feeding to enhance its transmission • Hematophagy: • Difficulty in ingesting a blood meal leads to more probing. Increased probing favors transmission of the parasite. • The blockage by the parasites interferes with feeding and limits the volume of blood a fly can obtain; this could explain why infected flies probe the skin more frequently and spend more time feeding. • Experiments showed that flies were more likely to transmit L. donovani to hamsters when they probed and took no blood compared to those infected flies that took a blood meal.

10. Transmission from the Vertebrate host • Host attractiveness is enhanced possibly due to modified host odor and vectors such as tsetse flies feed more frequently on infected hosts. • Host defensive behaviors may be reduced making feeding on infected hosts less risky. • Pathology associated with the infection could assist the blood-feeding efforts of vectors: • Thrombocytopenia induced by malaria, African trypanosomiasis and babesiosis decrease hemostasis.

11. Manipulation of insect vector by their parasite FECUNDITY, BLOOD FEEDING, AND INFECTION • Blood feeding is essential for both: vector and parasite. • Blood meal quantity and quality are important for egg production. • There is a reduction in fecundity (ability to reproduce) in plasmodium-infected mosquitoes, specially when oocysts are developed. • This loss of fecundity may be attributed to reduced blood intake or intake of impoverished blood when mosquitoes fed on infected hosts. • How could reduced fecundity benefit the parasite?Since increased reproductive effort decreases lifespan; if vector survival results from fecundity reduction then the parasite increases its chances of successful transmission.

12. Recommendations for vector control • Genetic manipulation of vectors: • Transgenic mosquitoes with impaired ability to transmit the parasite. This is attractive because is likely to be economically viable and relatively “low technology”. • Vector immunity and vector-parasite interactions: • Genome projects would help identify novel targets in the mosquito gut and salivary glands involved in digestion of the blood meal and host-parasite-vector interactions which could be used to develop vaccinesthat block the transmission of parasites and mosquito immune regulators or ‘smart sprays’ that disrupt the development of the parasite in the mosquito. • Vector behavior and other approaches to vector control: • Elucidating the molecular basis of many mosquito behavior may be an expensive research investment, but the simple traps and repellant devises anticipated from this research could be easily adopted in malaria-endemic countries

13. Genetic manipulation of vectors • Two broad categories: • Genetic modification of mosquito populations. Could be achieved by releasing transgenic mosquitoes carrying genes whose products impair pathogen development. • Population suppression: use of sterile insect technique (SIT) in conjunction with “release of insects carrying a dominant lethal” (RIDL). Insects are transformed with a transgene whose product suppresses offspring production, leading to a decrease of the vector population. A transgenic mosquito carrying a gene that confers resistance to the malaria parasite. The mosquito can be recognized as transgenic by the green fluorescence of the eye facets. http://news.mongabay.com/2007/0319-mosquitoes.html

14. Genetic manipulation of vectors • Controversial but attractive and potentially self-propagating. Many questions need to be addressed first about the feasibility and consequences of this approach. Serious issues are: reduced fitnessof modified vectors, the ecological impact of transgenic arthropods and the evolutionary consequences of their release. • In addition scientists need: • Methodologies to introduce foreign genes into vectors mosquitoes (e.g. transposable elements) • Promotores that can drive the expression of foreign genes in the correct tissues and at the appropriate times need to be characterized • Find “blocking gene products” capable of interfering with parasite development in the mosquito. Deleterious effects of these gene products on the mosquito should be considered and avoided. Laser scanner image of transgenic mosquito larvae. The white areas of high fluorescent intensity indicate a high level of transgene expression - particularly in the gut of the larvae The transformation technique involves the microinjection of the recombinant DNA into the posterior end of mosquito embryos (fresh laid eggs) prior to pole cell formation.

15. Genetic manipulation of vectors • Transgenic: A genetically modified organism (GMO): an organism whose genetic material has been altered using genetic engineering techniques. The Transgene is a segment of DNA containing a gene sequence that has been isolated from one organism and introduced into a different one. This DNA will be transcribed and translated. In transgenic mosquitoes the idea is to transfer genes that will alter the transmission of the malaria parasite. • Experiment using the gene for the Green Fluorescent Protein (EGFP). The gene was inserted into a plasmid and pre-blastoderm embryos were injected with it and an average of 29% of the injected ones survived. From these, 50% were fluorescent. Confocal fluorescence and transmission microphotographs of putative homozygous (left) transgenic larvae expressing EGFP compared with a wild-type mosquito (right).

16. Effector strategies that could inactivate malaria parasites within the mosquito CHITIN • One example of a Parasite-secreted protein: CHITINASE • Ookinetes secrete chitinasesthat facilitate their crossing of the peritrophic matrix. • Parasite strains carrying mutations or gene knockouts of chitinase are markedly impaired in their ability to form oocysts. • Feeding anti-chitinase antibodies to mosquitoes also impaired the development of oocysts. Ookinete (O) penetration of the chitin-containing peritrophic matrix (PM) of the mosquito midgut. The parasite is exiting the bloodmeal on the left, producing chitinase focally to disrupt the peritrophic matrix, en route to the microvilli of the midgut epithelial surface at the right. Trends in parasitology, (2001) 17:269

17. Genetic manipulation of vectors • The ookinete cross the midgut epithelium and differentiate into oocyst. The sporozoites cross the salivary gland epithelium. • Scientists identified a peptide (SM1 for salivary gland- and midgut-binding peptide 1) that binds specifically to the two epithelia: the distal lobes of the salivary glands and the lumenal surface of the midgut. SM1 inhibits crossing of the two epithelia by the parasites. • The idea is that if you can produce SM1 into the gut lumen with a blood meal the Plasmodium development would be blocked. • They created a synthetic gene AgCP[SM1]4.They used a promoter which is activated by a blood meal, and a signal sequence to drive secretion of the protein into the midgutlumen. The gene was inserted into a vector and transformed into the germ line of Anopheles stephensi. Nature (2002) 417:452.17 • The expression of the peptide produced a reduction in the number of oocysts formed (69-95% inhibition) and also these transgenic mosquitos had fewer sporozoites in their salivary glands Detection of AgCP[SM1]4 transgenic mosquitoes by transformation marker-mediated fluorescence. Top, a wild-type (non-transgenic) larva (middle) flanked by transgenic larvae viewed from the dorsal (top) or ventral (bottom) sides. Bottom, the head of a wild-type (left) and a transgenic (right) mosquito. The entire eye expresses GFP. http://www.genomenewsnetwork.org/articles/05_02/transgenic_mosquitoes.shtml

18. Population reduction: SIT and RIDL SIT: Sterile Insect technique: male insects are mass-reared, sterilized by irradiation and then released in large numbers in the infested areas in order to contribute to sterile mating with wild mosquitoes. RIDL: release of insects with a dominant lethal . RIDL uses males carrying female dominant lethal transgenes that can produce purely male offspring. In the laboratory, this strain is maintained by using a repressible system to control transgene expression; absence of the repressor from the insect diet in the field activates the lethal trait. This approach does not appear suitable for malaria control because of its susceptibility to immigration from outside the target area. Sterilized male flies are mass released across the target area by airplanes. The sexually sterile males, which outnumber the wild-type males, mate with wild-type females resulting in infertile mating events. This results in a decrease of the pest levels and, if continued over several generations, the potential eradication of the pest from the target area. Nature Biotechnology 23:433 SIT has been used successfully to eradicate tsetse flies from Burkina Faso, Tanzania, Nigeria and Zanzibar where it eradicated Glossina austeni from the 1600 km2 Unguja Island.

19. Population replacement A transgenic mosquito (Left) with green fluorescent eyes, and a non-transgenic mosquito (Right), with no eye fluorescence. The transgenic mosquito carries a gene that confers resistant to the malaria parasite. An important issue to consider before the release of genetically manipulated organisms is the fitness of the mosquitoes carrying the transgene. The transgenic insect needs to compete with the local populations to efficiently introgress the effector genes into the wild gene pool. Fitness is the relative success with which a genotype transmits its genes to the next generation. Survival and reproduction are the important components.

20. Insect immunity • This approach studies vector immune responses, and its effect on parasite transmission. • The idea is to develop vaccines that block parasite transmission and antimalarial agents that target vector immunity and parasite development. • Killing mechanisms: • Antimicrobial peptides:defensin and cecropin genes in A. gambiae. Highly induced by malaria infection • Melanotic encapsulation: Two mechanisms: Cellular encapsulation mediated by haemocytes that surround and attach to the microorganism to form a capsule that becomes melanized. Humoralencapsulation is the formation of a melanizedproteinaceous capsule around the microorganism without participation of haemocytes. • Phagocytosis:killing of microorganisms through engulfment and subsequent degradation by haemocytes. Middlepanel: Organs and cell types involved in Plasmodium interaction and immune response, and the parasites life cycle. Gametocytes (GC) enter the posterior midgut (PMG) through the anterior midgut (AMG), fertilize and develop into an ookinete (OK) that traverses the peritrophic matrix and midgut epithelium to form an oocyst (OC) under the basal lamina. After maturation, sporozoities (S) translocate from the oocyst (OC) to the salivary glands (SG). Lower panel: Melanized ookinetes in the midgut epithelium (MOK1, MOK2). Melanization is stronger on the apical end that is facing the haemolymph. Ookinetes (OK1, OK2) can be visualized in the midgut epithelium when stained with an ookinete surface protein-specific antibody. Intact mature oocyst on the basal side of the midgut epithelium (OC1). Cellular Microbiology (2003) 5, 3-14

21. Other research initiatives • Understanding vector biology • Mosquitoes show a remarkable preference for humans as hosts for blood-feeding • They are highly susceptible to infection • Olfaction plays a crucial role in shaping behaviors such as host seeking and feeding and determines their vectorial capacity. • Research on the behavior of vectors: • Modification of vector behavior for disease control • Basic research to understand the genetic and environmental components of vector behavior and reproductive biology • Mosquito genomics: comparative genomics to provide information about lineage-specific adaptations, population biology, ecology and genetics, dynamics, regulation and variation of vector populations, and vector survival strategies.

22. Other research initiatives Investigating mechanisms of insecticide resistance • At present, the control of malaria vectors relies extensively on the use of indoor house spraying with residual insecticides and the use of insecticide-impregnated bednets. • The problem is the lack of available licensed insecticides and the growing resistance. • Novel targets and the understanding of resistance are also important areas of research. • Scientists are trying to identify specific members of the detoxification enzymes whose expression are elevated in insecticide-resistant populations

23. SUMMARY • Remarkable progress has been made towards a the understanding of vector biology and the potential manipulation of its host for its own benefit (transmission and metacyclogenesis). • New tools are available and are being produced for the production of transgenic vectors. • Fitness of the transgenic population in the wild is a problem to be solved. • Mosquito control measures are often complicated by the presence of multiple vectors in the same area. In Africa, many different anopheline species can function as malaria vectors, either simultaneously or seasonally. • The A. gambiae genome has been completed so the number of neutral genetic markers for estimating gene glow between populations has increased. Strategies for malaria control