Method for the Reduction of mosquitoesvectors of West Nile virus and Dengue fever

Gerardo Ulíbarri, PhD

Sept. 2013

What are mosquitoes good for?

• Food for other animals/insects• As Larva or adulto mosquitoe

• Good virus/parasite wells• Vectors of viruses/ parasites

Some of the illnesses that mosquitoes can transmit to humans

• Yellow Fever (Aedes aegypti)• Dengue Fever (Aedes albopictus/aegypti)• Malaria (Anopheles gambiae)• West Nile virus (Culex pipiens)• Lymphatic filariasis (elephantiasis)• River blindness

Some of the illnesses that mosquitoes can transmit to animals

Encefalitis Equina del Este (Aedes vexans)Encefalitis Equina del Oeste (Culex/Culiseta)Malaria aviar (Culex quinquefasciatus)Virus del Oeste del Nilo (Culex spp)

3561 different species of mosquitoes around the world

http://www.mosquitocatalog.org/default.aspx

México 224 Continental United States 166 Canada 74

Walter Reed Biosystematics Unit (WRBU), Division of Entomology, Walter Reed Army Institute of Research (WRAIR), Silver Spring, Maryland, USA.

Anopheles spp. 478 Aedes / Ochlerotatus spp. 206 Culex spp. 789

México

Anopheles spp. 26 Aedes / Ochlerotatus spp. 25 Culex spp. 58

3561 different species of mosquitoes around the world

The mosquito reproductive cycle

• Mosquitoes undergo metamorphologicalchanges

• Only females need blood to develop their eggs

• Three days after biting, the female can lay from 3-300 eggs

• In 48 hours larva can emerge from the eggs

• Larva needs air to survive

• Adults emerge after 2 days in pupa state

http://commons.wikimedia.org/wiki/File:Culex_mosquito_life_cycle_es.svg

El ciclo de reproducción del mosco

• It needs from 4-6 days to go from egg to adult, dependent on Temperature

• Larva can survive in only 1 cm of water

• Eggs of some species can survive for years in drought

• Some eggs can support very low temperatures

• There are mosquitoes that prefer humans (antropophilic) to other animals for blood.

http://commons.wikimedia.org/wiki/File:Culex_mosquito_life_cycle_es.svg

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1) Biological

2) Physical

3) Chemical

Methods used to control or reduce mosquitoes

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1) Biological: Uses natural predators of the mosquito

Mosquito Fish (Gambusia affinis)

Bacteria: Bacillus thurigiensis israelensis (Bti)

Bacillus sphaericus

Dragon Fly

Genetically modified mosquitoes

Life history variation of invasive mosquitofish (Gambusia holbrooki) along a salinity gradient.  C Alcaraz, E Garcıa-Berthou. BIOLOGICAL CONSERVATION 139 (2007) 83–92.Bacillus sphaericus as a mosquito pathogen: properties of the organism and its toxins. P Baumann, M A Clark, L Baumann, and A H Broadwell. Microbiol Rev. 1991 September; 55(3): 425–436.

Methods used to control or reduce mosquitoes

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Insects and animals that feed on mosquitoes

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1) Physical: Layer of oil in water surface

Water container coverts

Adult Traps

Carbon dioxide, water steam and heat

Photonic fence (micro laser)

Lethal Ovitraps (use insecticide)

Bed nets

Clean Patio

Methods used to control or reduce mosquitoes

13http://www.educasocial.com.uy/demo/mod/file/download.php?file_guid=1039

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1) Chemistry: pesticides Organochlorinated -DDT Organo-phosphates Carbamates Piretotroids

Neurotoxicity: prolonged activation of Sodium channels causing depolarization sustained depolarization in neurones, producing muscular spams.Teratogenicity: abnormal fetus developmentDDT: Inhibits shell formation in bird eggs.

Permetrine

Malathion

Carbaryldichlorodiphenyltrichloroethane

Methods used to control or reduce mosquitoes

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Methods used to monitor the presence of mosquitoes vectors of disease

How is it determined when is the right moment to spray pesticides

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CDC Light traps(Centers for Disease Control and Prevention)

(www.cdc.gov/)

CDC Light Traps

•Traps only adultmosquites and other inects

•Mainly females•Uses light and CO2 toattract the insects

•Problems•There is no selectivity

•High cost of operation•~$15 dollars per night /per trap

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Larval deep

www.keysmosquito.org/larvicides_treatments.html

www.ocvcd.org/mosquitoes2.php

•Problems•Every pond ahs to be verified independently

•High operative cost (labor)

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Human Bait

news.cals.wisc.edu/newsDisplay.asp?id=1771

www.trevorwilliams.info/Mosquitoes_blackflies...

•Problems•Only anthropophagic mosquitoes are trapped

•Dilemma with the ethic of using humans as bait

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Gravid y Ovitraps

www.ocvcd.org/mosquitoes2.php

www.fehd.gov.hk/.../2002/calendar-photo5.html

•Problems•Very selective. Attraction depends on solution used•Only attracts gravid females

•Problems•Very selective, Attraction depends on the solution used•Only collects eggs/larvae

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West Nile virus-The North America Problem

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Culex spp – Main vectors of the West Nile virus

http://www.madrimasd.org/blogs/virusemergentes/2012/10/el-avance-de-los-flavivirus-emergentes-y-reemergentes/

22www.usgs.gov/125/articles/images/wnv_map.jpg

http://www.cdc.gov/westnile/resources/pdfs/cummulative/99_2012_CasesAndDeathsClinicalPresentationHumanCases.pdf

West Nile virus infections

1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012US 62 21 66 4156 9862 2539 3000 4269 3630 1356 720 1021 712 1590Canada 414 1481 25 225 151 2215 36 13 5 101 62

0100020003000400050006000700080009000

10000

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Mosquito BehaviourMosquito behaviour is mainly influenced by a series of olfactory stimuli.

W Takken, and B G Knols. (1999). Annu. Rev. Entomol. 44, 131-157.

Odorant Binding Proteins (OBP)

• Water-soluble globular proteins with exposed hydrophilic groups

• Located in the lymphatic space surrounding the sensory neurons

• Transport the hydrophobic odorant molecules through the aqueous lymph to the odorant receptors.

Proteina tipo-G acoplada a receptores (G-PCR)

• Seven transmembranehelical proteins

• Residing on the dendritic membrane of the olfactory sensory neurons

• Activated when bound to OBP which in turn activates the olfaction signal

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Receptor del olor 7 (Or‐7)• Identificado y caracterizado en varias especies de

mosquitos: Anopheles gambiae (AgOr7), Aedes aegypti (AaOr7), Culex quinquefasciatus (CqOr7)

• CqOr7 fue identificado en Marzo de 2006 por el equipo del Dr. Zwiebel, el mismo grupo que secuencio el genoma del mosco Anofeles. (Xia and Zwiebel, 2006).

Xia, Y. & Zwiebel, L. J. Insect Biochem Mol Biol 36, 169‐76 (2006)

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Modified Ovitrap

http://www.youtube.com/watch?v=VwIqGbhq4T8Mosquito laying eggs, eggs hatching (#311) nature1upclose·

Bacteral fermentation

H2O

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Culex egg rafts on modified ovitrap

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www.qac.org

Egg Raft

Larva

Pupa

Modified Ovitrap

Field studies

We have found up to 89 rafts in one night study(~26,000 eggs)

What is the secret?

The apical part (superior) of each egg contains a chemical compound which serves a signaling to oviposition to new comers (attractant) for the Culex spp.

Initially, the mosquitoes are attracted to the ovitrap by the natural concoction.

3434

Sudbury

Ovitrap

Day Temperature (oC) Relative Humidity (%) # of Egg Raft / Day

Max. Mean Min.

1 24.4 18.45 12.5 87.42 41

2 27.9 22.15 16.4 73.96 146

3 30.3 24 17.7 70.88 52

4 31.6 25.05 18.5 59.54 67

5 31.4 24.85 18.3 66.21 33

6 31.4 25.05 18.7 62.08 30

14 29.4 22.9 16.4 90.25 47

15 22.8 19.7 16.6 70.58 30

16 25.5 21.7 17.9 91.38 103

17 29.3 23.2 17.1 77.29 116

18 21.7 18.3 14.9 70.21 165

19 22.2 17.85 13.5 80.33 65

20 27.5 22.8 18.1 89.88 52

21 33.2 27.2 21.2 81.38 119

Total 2007

Average 91

Sudbury

Ovitrap

Average eggs in a raft (Sudbury)= 270 (Culex pipiens/restuans)

In the first field study 2007 rafts werecollected in 21 days = 541,890 eggs

An average of 25,804 eggs per day

Using only 3 modified ovitraps

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2007 Field study150 modified ovitraps were used in 12 different sitesField study was run from 1st of June to the 30 of August

• 11196 rafts were colected in 90 days• 870 rafts per week• 3,358,800 eggs destroyed!

If one estiamtes a 20% of the eggs hatching producing females capable of laying eggs;

In the first two weeks they will produce:(870)(2)(270)=469,800 x 0.2= 93,960 females

If each lays eggs once; 25,369,200 eggs potentially produced

In Only Two weeks

But, does this really cause a reduction of mosquitoes?

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NCDC Light Trap

Artificial Breeding Pools4

CDC Light Trap

Ovitrampa modificada4 1

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1 2 3 4 5 6 7 8 9 10Sudbury control 1 9 4 24 10 78 74 89 52 44Long Lake 2 1 3 5 1 2 2 0 0 0

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Adu

lt C

ulex

mos

quito

es

per

trap

ping

eve

ntComparative study between a test site using modified ovitraps and two 

control sites without ovitraps, in Sudbury

Collection of adult mosquitoes was done using the standard CDC light traps on each site

Ovitrap

ABP

Bacterialfermentation

Rafts

Dumpsolution

Bacterialfermentation

Rafts

Filtersolution

Time

Time

Attr

activ

enes

sA

ttrac

tiven

ess

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fehd.gov.hk

OOH

O

O

erythro‐6‐Acetoxy‐5‐hexadecanolideB.R. Laurence and J.A. Pickett J. Chem. Soc, Chem. Commun. 1982 p. 59-60

Do the modified ovitraps work as well with other species?

A female mosquito (Anopheles gambiae) feedingPhoto courtesy Centers for Disease Control & Prevention, photographer Jim Gathany(http://home.howstuffworks.com/mosquito-magnet1.htm)

Aedes aegypti

(http://en.wikipedia.org/wiki/Aedes)

http://www.paho.org/hq/index.php?option=com_content&view=article&id=264&Itemid=363&lang=en

Dengue Regional Information: Number of Cases-2013

Estado del estudio en Pentatlán, Gro, México.Inicio : primera semana de Febrero hasta Ultima semana de Agosto 2013

(30 semanas de estudio)

Modified Ovitrampa

Aedes eggs on a paper pellon

Photo of Aedes mosquitoe eggs on paper pellon

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Atrayente

Control

Total number of Aedes mosquito eggs collected during the field study in Petatlán, Gro

Semana 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16Atrayente 4575 6643 7076 6515 5617 4502 4001 5744 8870 7570 4769 2622 2425 3887 4653 4664Control 1048 481 785 665 776 896 503 602 704 1180 494 368 553 668 430 606

Huevo destruido 5623 7124 7861 7180 6393 5398 4504 6346 9574 8750 5263 2990 2978 4555 5083 527017 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

3763 3005 2695 3552 2802 4799 4134 4640 4921 5217 4599 4184 4763 3959 7304 7119723 621 690 713 624 806 820 876 910 1151 1094 808 855 889 1013 12204486 3626 3385 4265 3426 5605 4954 5516 5831 6368 5693 4992 5618 4848 8317 8339

Atractant= 40 ovitrampasControl = 10 ovitrampas

Total 155,58924,572180,161

Week

Total eggs collected

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Control/ovitrap

Attractant/ovitrap

Semana 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Atrayente/ovitrampa 131 190 202 186 160 129 108 144 222 189 119 66 61 97 116 117

Control/ovitrampa 70 32 52 44 52 60 75 105 70 118 49 37 55 67 43 61

Semana 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

Atrayente/ovitrampa 94 75 67 89 70 120 103 116 123 130 115 105 119 99 183 178

Control/ovitrampa 72 62 69 71 62 81 82 88 91 115 109 81 86 89 101 122

Average eggs collected per ovirapduring the study (32 weeks)

Attractant 126

Control 74

Eggs collected

 per ovitrap

Week

Total number of Aedes mosquito eggs collected during the field study in Petatlán, Gro

In Red-Egg couunt using traditional ovitraps with attractant solution (Downtown Petatlan) In Blue-egg count using traditional ovitraps, where the modified ovitraps were located.

Semana/mesTerceraJunio

CuartaJunio

PrimeraJulio

SegundaJulio

TerceraJulio

CuartaJulio

PrimeraAgosto

SegundaAgosto

TerceraAgosto

CuartaAgosto

Quinta Agosto

Control / ovitrampaEn lugar del estudio 18 3 0 20 13 17 11 2 8 18 12Atrayente / ovitrampa

En lugar ajeno al estudio 29 79 104 97 71 63 37

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120

Control / ovitrampa en lugar del estudio

Atrayente / ovitrampa en lugar ajeno al estudio

Week

Eggs collected

 per ovitrap

Comparative Field studies between Petatlán, Gro, México in 2013 (Green)and a field study in Zayaxche, Guatemala en 2010 (Blue). Observe that the count does not go up during the rainy season in Mexico (July-Sept).

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E F M A M J J A S O N D

Atrayente/ovitrampa‐Gua Control/ovitrampa‐Gua Atrayente/ovitrampa‐Pet Control/ovitrampa‐Pet

E F M A M J J A S O N D

Atrayente/ovitrampa‐Gua 71 273 308 877 744 1135 1555 371 350 300 256 240Control/ovitrampa‐Gua 37 109 172 276 137 245 407 164 205 206 113 103Atrayente/ovitrampa‐Pet 709 568 596 485 231 462 568Control/ovitrampa‐Pet 199 291 60 298 202 341 480

Conclusion: • The modified ovitraps reduce the presence of the adult Culex spp. mosquito vector of the

West Nile virus in above 80% when a Culex-specific attractant solution is used. The solution that has been used is based on a known bacterial/yeast fermentation of natural plants and a few other attractant ingredients.

• The modified ovitraps maintain low the population of the Aedes spp. mosquitoes, vectors of the dengue virus, even during the rainy season. The difference of adult mosquitoes between the site with modified ovitraps and a site with no ovitraps is approx. of 60-70% (to be verified). The attractant solution has been develop exclusively for the Aedes type of mosquitoes.

• Modified ovitraps are being studied with different attractant solution in Zayaxche, Peten, Guatemala against the malaria mosquitoes by the team of Jacobo Rojas y ValentínSalazar from the local Health Unit.