Host seeking interrupted: behavioural analysis of mosquito responses to bed

nets

J.F. SutcliffeDept. Biology

Blood feeding and disease transmission

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over 20 arthropod groups have evolved blood feeding behaviour- these include several “true fly”

(Diptera) families, for example:Simuliidae – black flies

Bioimages - UKCulicidae - mosquitoes

USDA

Ceratopogonidae – noseeums

Inst. Animal Health - UK

Psychodidae – sand flies

Univ. Keele - UK

Haematophagous

micropredators–

stalk and locate blood hosts using breath and body odours, heat, moisture and vision- feed on small amount of host tissue (blood)and escape- feed again within a few days on a new host- insect “vectors”

provide an excellent means of transmission for many blood-borne pathogens, e.g., yellow feverdengue, bubonic plague, typhus, malaria…

Mosquitoes –

the world’s most dangerous animal?

http://www.gatesnotes.com/Health/Most-Lethal-Animal-Mosquito-Week

Almost all of thesedue to malaria

Mother and child sleeping in an LLIN

WHO/TDR/Crump

Damaged bed net

Courtesy S. Smith, CDC Entomology Branch

Bed netsLong lasting insecticide treated bed nets (LLINs) are a major strategy for combatting

malaria

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hundreds of millions of LLINs

have been distributed as part of malaria reduction programs such as the CDC-managed President’s Malaria Initiative (PMI) - over time LLINs

deteriorate physically and lose insecticidal effect-

cost-effective decisions that protect public health must be made about when to replace damaged nets but there are currently no evidence-based methods determine how much damage is too much (= net failure)

Net

DawaPlus

DuranetInterceptor

NetProtect

OlysetPermaNet 2.0

PermaNet 3.0

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60

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Mean number of holes per net after 36 monthsKenya

Bed nets work because

Anopheles mosquitoes bite and transmit malaria mainly at night

CDC Public Health Image Bank

#*&%#$^

Host seeking mosquitoes respond to host stimuli and their environment to locate their hosts

Host seekers face many obstacles and vagaries on way to host and

often fail to find host and get blood meal

Bed nets are just one more obstacle faced by the host seeking mosquito. How do they respond to this obstacle?

Mosquito research at the Centers for DiseasesControl in Atlanta, Georgia

Extensive insectary

facilities includeinclude climate controlled rooms forlarge scale experiments and manymosquito species in colony

Three questions addressed:

Question 1) How likely are mosquitoes to go through holes of different sizes and shapes in the net?

Question 2) What parts of the occupied bed net are under the greatest ‘mosquitopressure’

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where are the mosquitoes most likely to try to get in?

Question 3) How can we combined these findings into a model predicting vulner-ability of damaged bed nets?

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Mosquitoes fly/skim close to the net surface when searching for holes

search occurs largely in two-dimensions

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Searching mosquitoes may come across holes in the net but not pass through

hole passage

must be preceded by hole encounter

but doesn’tnecessarily follow it

Therefore, the probability (P) of a mosquito entering net through a hole = P(encounter) X P(hole

passage)

Question 1) How likely are mosquitoes to pass through holes of different sizes and shapes in the net?

Lessons learned from previous observations

Experimental systemFibreglass

screen “bed net”

system in3mX3m containment tent:- smooth flat surfaces for video-

interchangeable system for switching different holes in and out-

fibreglass

screen accommodates various sizes and shapes of holes in material that does not ravel- experiments done in darkened room

3) 30cm X 30cmsampled area

1) position line laser to project along outside about 1 cm away from net to capture mosquitoes when against the net2) position another line laser to project close to the inside of

net to capture mosquitoes as they pass through the hole 3) define a 30cm X 30cm sampling area around pre-cut net hole (corresponds to 30cm X 30cm sampling area of sticky squares) 4) place camera(s) to record activity on net near hole(s)

Configuring the fibreglass

net for video observations

1) green laser source outside

net2) red laser source inside

net

fibreglass

screeningin aluminum framesforms walls and roof

4

1cm 1cm approx. 1cm

inside the net

outside the net

red laserfield

green laserfield

bed net

edges of net hole

Net seen end-on

Observations1. Appearance 2. Encounter with hole

3. Net entry

line laserprojects along

net front

line laserprojects across

net roof

Laser fields withnet between.

Mosquito inside net

Various hole sizes/shapes

tested

Example of video observationsRight -

12mm X 200mm hole in side of net (taped over before experiment to prevent premature mosquito entry)

Below, left and right -

Screen grabs of experimental video illustrating hole (outline) and mosquito: 1) “appearance”(two mosquitoes), 2a) “encountering”

hole and 2b) “entering”

hole

1

2a

2b

What hole feature best predicts encounter probability?

- various candidates-

hole area-

hole width-

hole circumference

- hole circumference is the bestpredictor of hole encounteraccounting for almost 75% ofvariation in dependent variable

What hole feature best predicts hole entry

probability?

hole width, irrespective of other hole measures (e.g. length, area, circumference, etc.), predicts almost 83% of the variability in per encounter entry probability

0

90

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360

450

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0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96

18‐20

16‐18

14‐16

12‐14

10‐12

8‐10

6‐8

4‐6

2‐4

0‐2

Hole width (mm)

Per

mos

quit

o P

of g

etti

ng in

to n

et

Hole cir

cumfer

ence (

mm)

0.18-0.200.16-0.180.14-0.160.12-0.140.10-0.120.08-0.100.06-0.080.04-0.060.02-0.040.00-0.02

0.20

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0.08

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0.00

Estimating P(of

getting into the net) through holesof different sizes (estimating hole “passability”)e.g. 1) for 12mm wide X 150mm circum. hole, P = 2-4%

2) for 30mm wide X 220mm circum. hole, P = 4-6% 3) for 90mm wide X 360mm circum. hole, P = 10-12% 4), 5), 6)…

How many mosquitoes actually enter the net also depends on whether mosquitoes are found on the parts of the net where the holes are

Question 2

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6

4

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3

Question 2) What parts of the occupied bed net are under the greatest ‘mosquito pressure’

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where are the mosquitoes

most likely to try to get in?

All areas more or less the same?

Question 2) What parts of the occupied bed net are under the greatest ‘mosquito pressure’

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where are the mosquitoes

most likely to try to get in?

To the feet (smelly socks effect) or to the head (exhaled breath

effect)?

Question 2) What parts of the occupied bed net are under the greatest ‘mosquito pressure’

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where are the mosquitoes

most likely to try to get in?

Or do they follow a heavier-than-air-CO2

plume?

10’

by 10’

REI containment tent and bed net

Bed net with sticky screen squares

Bed net divided into 18 sampling areas

Experimental set up

Containment tent(3mX3mX2.5m)

Bed net (1.8mX1.3mX1.5m) on

frame hung with sticky squares (not shown)

Subject in bed net

Experimental set up (cont’d)

Up to 200 hungry female An. gambiae mosquitoes from CDC colony released into tent

Experimental set up (cont’d)

Subject exits after 2h, mosquitoes on sticky squares counted

Experimental set up (cont’d)

An. gambiae G3 females stuck to squares on top of bed net

J

Data presentation –

bed net folded out and flattened pictorially to depict results as a bubble chart

0.01

0.01 0.01 0.00

0.000.01

0.02 0.01 0.01

0.010.08

0.09 0.08 0.08

0.110.04 0.42 0.01A

NML

IHG

DCB

OJERQFK P

Generic layout of whole net capture dataEach lettered square corresponds to asector on net

end (head) top

side

end (feet)

Anopheles gambiae –

untreated net, subject A

1

10

100

Catches group into three zones

Hypothesized mosquito movement pattern around an occupied bed net (human subject not shown).

Sutcliffe and Yin Malaria Journal 2014 13:294

‘X’

–

mosquitoes re-leased‘A’

–

fly along floor, hit net, fly up‘B’

–

encounter hostodour plume, dropdown to roof, andto floor‘C’

–

fly along floor,hit net or tentsides, fly up again‘D’

–

encounterHost plume, etc.

Question 3) How can we combine these findings into a model predicting vulnerability of damaged bed nets?

Use:1) passabilities

based on hole width and circumference:Hole 1) 12mm wide X 150mm circum. hole, P = 2-4% Hole 2) 30mm wide X 220mm circum. hole, P = 4-6%Hole 3) 90mm wide X 360mm circum. hole, P = 10-12%

and2) mosquito densities

mosquitoes measured with sticky squares on the net:

to run some simple hypothetical scenarios

Number of mosquitoes entering the net per unit time = Hole passability

X Density of mosquitoes near the hole

(totalled for all holes in the net)

1

10

100

1.0 –

1.2

2.0 –

2.4Total =

Relative numbers of mosquitoes entering net/hour (assuming 200 present)

Net scenario A –

two hole 3s (90mm wide X 360mm circum.)in upper two thirds of side

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surprisingly few mosquitoes enter despite two large holes in net

1.0 –

1.2

Mosquitoes entering

20.0 –

40.0

1.0 –

1.2

4.0 –

6.0

25.0 –

47.2Total =

Relative numbers of mosquitoes entering net/hour (assuming 200 present)

Net scenario B –

size 1 hole in roof, size 2 hole in bottom third of side,size 3 hole upper third of side

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smallest hole contributes over 80% of mosquitoes because of its

locationon the roof- largest hole contributes less than 5% of mosquitoes because its

in a location where mosquitoes are sparsest

Mosquitoes entering

Conclusions/recommendations

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even one small hole on the net roof may render a bed net non-protective

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nets with large holes may still offer protection depending on where the holesare located and the overall density of mosquitoes

- net vulnerability can be relatively easily estimated by collecting informationabout:

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number of holes-

locations of holes - widths of holes-

circumferences of holes

Part of current protocols?YesNoNoNo

Now the scientific evidence must find its way into policy…