METHODOLOGY

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Majority of the studies were conducted in the field so as to

obtain a true picture of the behaviooral pattern of ~ i o i ~ .

Field studies included observatims on the mo~qvito fauna of

Shcrtallai region, seasonal prevalence of Mamonioides, adult

emeqanco pstter.1, reproductive behaviourr resting behaviour, biting

bahaviaur etc. Studies such a8 determination of the duration of

goootrophic cycles, emergence periodicityr wipoaition rhythm and

susceptibility status ot rtansonioides to 8. malayi infection were - nabe under laboratory conditions. Susceptibility status of

Mnacmioides to different groups of insecticih was also

invest iqated.

4.1. Mult Ecology:

4.1.1. Field studies:

4 .l. 1.1. Adult mergence pattern of Hansonioides:

Iha bnsdinq habitats of Manacmioides are water bodies infested

with different varieties of -tic vsgetation (hydrophytss). Rn

prwalmm and distribution of all the available water bodice

infeatrd with hydrophytea mra surveyed in the taluk, for a period of

ar(, ymr. Statiatica ragardfng fallw lsnds and psk)ll fielcb -re

obtained fran the 'Krishi Bhavans' (Department, of Agriculture, Gout.

of Kerala), located in each 'panchayat'. Eawrgence trap were Set up

on all types habitats, at fortnightly intervals in different reqion8

of the taluk to determine the adult emergence potential. The

emergence traps (Plate I ) designed for this purpose uere stitched out

of ~wquito-proof (17 to 18 meshes per an) nylon cloth in the shape 2

of a bed net structure, with a surface area of 4 m . To the f a x upper corners of the trap sleeves were stitched so as to facilitate

collection of mosquitoes trapped inside, using an aspirator tube.

Sixteen steel rinqs uere fastened to the bottom portion of the trap

through which ran a nylon thread w that by pulling both ends of the

thread, the bottom portion can be cloaed. 'Lhese traps were suspenlkd

by ropes w e r the breeding habitats keeping one-fourth of the height

fran the bottan portion imnersed in water. FLnergence trap were set

up around 9.00 An and the emerging adults trapped inside were

collected after carefully entering inside the trap using aspirator

tubes on the rmbsequent day at about the same hour. In deep habitats

bottan port ion of the trap vas closad by pulling the threads, removed

fran the habitat and the moequitocrs trapped inside uere collected.

*

In pddy fielda only inmatures were sunteyed as emergence trap

collectia~ m y m e the crops. In Verntranattu lake both imnature

samplings wd trap collections were carried out. The oarparati* role

of different typas of b d i n g habitats on the adult d l ~ ~ q e n c e

potmtial of H. artnulifera, M. uniformis and H. indiwa ws

determined, unify analysis of variance test I A N O Y A ) . Factorial

analyain uan also m;l& to determine the rnle of different factors on

the mnerqence potential of Mansonioides ('Ibckey Test).

Tatal emergence rate of Mansonioides moquitoes (species-wise)

for the whole taluk wan comp~ted from the data on the prevalence of

different types of breedinq habitats in the taluk and the average

daily enrsrgmce patential of Wnsanioi&s, determined from these

habitats wing emerqence traps. Ihe sex ratio of Mansonioides

maquitwe from these collections was also analysed.

Sueepnet ml lect ims were h e during the dusk and dam hmrs to

trap the suannlnq moxplitoes. Cbaervations uere made in niqht biting

onc.1 restinq collectioru for mating soecimens as it has been reported

that mat inq occurs in the vicinity of hosts (Jayawickrema, 1953). The

propart ion of rmle specimens obtained in niqht restino collections in

cattle she& 1a.s recorded. The female m q ~ i t m s t r a m in the

anerqrwrce t.rapt were di-uected in the laboratory for their

inneminat ion stat uir .

4.1.1.3. Biting Collections:

Night bit inq collect ions were mducted overniqht to determine

Page . . .20

t h e degree o f man v e c t o r c o n t a c t th rough d i f f e r e n t seasans i n seven

f i x e d c a t c h i n g s t a t i o n s s p r e a d wer t h e *ole t a l u k (Armr,

Kuntpankulangara , Areeparanbu and t larar ikulam s o u t h i n t h e western

p o r t i o n , S h e r t a l l a i tom i n the c e n t r a l r e g i o n and Muhamna and

P s l l i p u r a ~ i n the eastern p o r t i o n ) a t an i n t e r v a l o f one month f o r a

p.riod of two years (1986-1988). As mst o f t h e areas were covered

udrsr a cactrol progrmme i n 1988 t h e d a t a f r a n one s t a t i o n , v i z .

Phrarikulm s o u t h (check zone) alone, were used u p t o 1990. B i t i n g

collections uern made u s i n g a human bait who was a l lowed to l i e d z m

i n thc verandah of t h e haubc keeping h i s anns and leqs bare.

nbsguitac?s prcb inq to bite were collected w i n g torches and test

tuben ( P l a t e XI).

S i u u l t a n e a u s i y , b i t i n q collection8 were made both i n s i d e and

o u t s i d e (dxut 15 m m a y ) of a house, so as t o d e t e r m i n e t h e k r e e

of cmb-exoClhbqy of tbn,wclioides. ?he* c o l l e c t i o n s Here conducted

for ane y e a r , at m i n t e r v a l of one m t h i n an a r e a ( - S h e r t a l l a i

toul).

B i t i n q collections were a l s o conducted ~ i n u l t a n e o u s l y u s i n q

c a t t l e Md man b a i t s fran 18.00 t o 22.00 h o u r s , to d e t e r m i n e t h e

h o s t pref crmm .

A l l t h e m c m p i t o e s o b t a i n e d vere brought to l a b o r a t o r y ,

i h t i f i e d and t h e tlansonioides mosquitoes were dissected f o r p a r i t y

Page . . .21

status end filarial infection.

Thn periodicity in the biting behaviour of Fhnsclnioides in

different biotopes (verandah, indoor and outdoor) uere analysed fran

the msber of arosquitoes collected during different hours fran man

biting collections. For the verandah, the data obtained from 172

mllectiona (1986 - 1990) uere used. Ihe man biting densities,

hourrise were oarplted M William's M n (W) expressed in

percentsges \Irhich gave the best measures of central tendency (Haddov,

1960). Since t l ~ e tW is a modif led qeanetric mmn, it also a1 l o w zero

values, i f any in the data. Hour-wise percentwe distribution ( W ) of

biting density and parity statw of Msonioides uere plotted in

graphs, .species-uise. The parity status in relation to the rhythmic

activity of bitinq ua8 not analywd for - M. indiana, the scwple size

bing -11. Ihe bitinq periadiclty of 13amioides in indoor and

outdoor MI alrro snalysed follcwinq the .mme procedure. Ihe relat iue

transnibaim potential of the vectors throuqh time was calculated,

basad an the data abtsined on msn vector contact.

4.1.1.4. r(egoon Trap Collections:

A Hagoar trap was sat up in tlnrarikulam South. The trap w a s

amatructed follauing Sewin (19761, with certain modifications. The

k.. of tho trap uaa8ur.d 1.25 n X 2.00 m with cno of the tuo largrr

uptight aid.. 2.20 m high j o i M by a aloging and water-proof roof to

Page . . .22

the opposite vertical sides which is 1.70 m high. A & o r was fitted

at om end of the trap. The bottom portion (0.60 m high from the

floor) of the eides of the trap and the door wae m d e up of wooden

planks 1.25 aa thick sbavcr which a horizontal entrance elit which

extended caaplaely round the trap including the door. It was made

frm 10.00 an wide moden plank placed to form a ' V ' shaped trough

with 20.00 cm vide opening to the outside converging to leave a 1.75

an slit like opening into the trap. Ihe wall of the trap above this

emtrancc slit was nradt up of mquito proof double nylon net, up to

roof.

A calf wan tied inside the nragoan trap in the evening and the

mquitocrs trapped inside were collected in the n e x t day morning.

Thasc mllecticma uere mncbcted for a period of one year (1988-1989)

at lrnnthly intervals. Bitinq collecticms usinq mm as bait uere also

conducted fran 18.00 to 06.00 hours in the verandah of the house, '1

d]acmt to the mqmn trap for ~amparisa?.

4.1.1.5. Rcratinq collections:

FIlretinq nrosguitocs r e smpled in all the pcmsible habitats

such ss h u ~ n thllinqs, cattle shcds and outdoor (shrub8 wd

bushrcs), rnployinq di f farsnt nrcthods as &scribed belau.

tnkor nstinq crollsctions *re conducted in all the mven areas

oelectsd for ssmpling the biting amlation. In each area 18 hduses

vkra chasen m fixed catchinq stations. Tvo people ainultaneously

marched inside these human c)vellinqs with the help of a flash light

war the wallst roofs d hanging objects for ten minutes in each

hcuee rrnd tho reat ing m a q i toes were collected using an aspirator

tube (Plate I . Ihc, marches were ma& between 08.00 and 09.00

hours by six people si~ultanmualy~ maintaining an uniform period of

air aan-burn. 'IhQ, maquitoas obtained were transferred to are foo t

Bar rad cage and bmqht to laborstory for identification. lhese

collsctim were carried out at monthly intervals, for a period ~f

tw yosra (1986-'88).

Ihe nst ing pgulatim of Nansonioides in day and night hours

wre Bctamimd by corrhrcting indoor resting cx>llcctions follwinq

the osual pw;.adurc~ at 08.00 to 09.00 hr I6 arw hours) and 18.00 to

21.00 hrs. respectively. During the night hours two m-hours "f l~e

m t for wrplinq reatinq laoeguitoes in es& hour. W areas viz.,

Mararikular north rwd hh&lma were selected for this purpose. Ihe

inibot mating &nsitiea recorded during day and night were subjected

to statistical malyuis (student ' t ' test 1 for determining whether

there w e any aiqnif icant differences. m e aWaninal conditions of

moup~itoclz, in day and different hours of the niqht also were

eubjoct.6 to statirtical analysis, to vrdcrstand the house

traqwnt in9 khsviour of Mmaonioides.

Page . . .24

Total catches using pyrethrum spray sheet collections were

roclductad r a d m l y in the above tuo stations both in day and night

follarcd by indoor resting collections. 0.04% pyrethrum in kerosene

warn qxayed inside the huts after closing all the doota and win&ws

cnd crwiccs. For each hut, ap~roximately 200 ml of this mlutim ma

sprayed ea a fine mist, l y l r q a hand sprayer. After 10 &esr all

the mosquitoser knocked down on the uhite sheetsr spread over the

f l m r -re picked up by forceps vith care. The rnosquitoea were

identified and dissected in the laboratory. % mean hut density

obtained cm total catches uas carpared with the indoor resting

dQMity of esch hame to deternine the hand catch efficiency.

Cattle ah& meting cbllectiars were eardocted in six fixed

stat ions, clpenfinq two mnn b r a in each station, between 08.00 and

09.00 hr osinq a fl& light snd aspirator. The cattle shed meting

pgxltst ion in night burs uss alao sampled follcrvinq the @hod

adopted in the huMn duellinqs. ?he results ere subjected to

stat i s t ica l analysis.

aJtdaor resting collections were done uaing aveeprets and

pir rat or tubw in b u s h and shlzlbs for a period of m e year in two

stat i o ~ v i z . , Mdrwnvs and Kurupankulangara, spcnding four man hclucs

each ti- at sn interval of am month. Draprst cages *re a l w wed

t o -1. th., maquitolbs reat inq artdoor (bushes and shrubs). Ihe

wad for thio plrpon were a modifi.d one (Plat* I V ) d.ecrib.cl

Page ... 27

4.1.2.2. Corrotrophic Cycle:

Iha &rat im of first and subsequent goslotrophic cycles were

Qtennined for H. - annulifera, using freshly emerged specimens reared

in a colony a g e by offering chicken blcd wery night. Witrape with

the favoured host plant, - P. strat iotes were kept inside the colony cage. ?he total m&er of egg clusters laid and the ntrmber of eggs in

ssch cluster were recorded daily in the morninq hours. Ihe

diettihut ion through days was plotted in a graph to determine the

peaks of aviposition, so 8s to elucidate the duration of first and

m&eqwmt qonotrophic cycles. The teneral stages of the first

qaratrdphic cycle were determined by dissectinq a sanple of

lnosquitoea daily cmmncing fran the subsequent day of first blood

nrml and by ncordinq the Sel)sls (1920) abdaninal conditions and

Chrietgrher '8 ( 1936) wariolar atages.

Pariodicity in the wipoerition activity of H. - wnulifera was

abserved in colony caqes. Iho nunt>er of sgg clusters laid in witraps

with E. stratiotes was recorded at an interval of three hours round

th. clock. Ihe result8 wore exproc~drd a8 'IW'in percentages Md

l e i p u d in a qraph, to determine t b rhyttmic activity of

wip#itiar.

4.1.2.4. Sosceptibility statw of vectors to different group of

inscrct i cides:

Picld collected fullfed spccimeoa of M. - uniformis were subjected to suaceptibflity testa for different doses of DLYI', BHC and Malathion

follwinq the standard procedures (WHO., 1981).

Paprlat ion Dynamics:

4.2.1. Fecundity:

'me rtwber of eoqs laid in the first and the subsequent

gonotrophic cycles was determined in the colony cages maintained in

the laboratory, usinq field collected freshly emerqed specimens of

n. annulifera by offrring chicken blood feed, through different - Wkbons.

Adult survival and lcsnqevfty:

b i l y &It mrvival rates of E. annulifera, thmugh different

m t h s were calmlatad fran perity status of the specimam cbtained

frm indoor rc.lrtinq collwtions using the following fornula of

thore n = tho &+@tion of ponattophic eyele rd P - pcaQattion p r a m *

Page . . .29

me Qily survival rates were also romprted f r m infection and

infectivity status using Mc Donald's formula, modified by Whartan

(1967) an fa1 lms:

tog ratio

L o g p t ------------- ( n - 4 )

Wwre Loq ratio is the ratio of mature larva rate to total

f i lar ia larva infection rate, P = propartion of d a i l y survival and

n = duration of extrinsic cycle.

Altemtively, the daily adult survival rates were c a l ~ l a t e d

edopring Laurence (1%2) method as shacrn below:

uhere p = proportion of daily adult survival, n = duration of extrinsic cycle and I = praporticm infective to total infected.

Ttrc adult langwity was determined in colony cagej maintained in

the laboratory, expossd to normal atlros@wric conditima.

4.2.3. R.productive potential:

R.probuctivo potential of E. annulifera wu araprt.6 for

Page ...m

different mtbr by reckoning life and fertility tables following

the method propad by Birch (1954) and modified by Sauthvooe (19661,

Cueller (1%9) and Rajagq#lan et a1 . , (1977 a). --

The Net reproductive rate (R or ntrmber of times a pqmlatim 0

could mltiply per generation in the absence of mortality was

Mere ' x ' = bration (age) in days, 'lx' = the probability of

the rrotvival at time ' x ' and 'mx' = the n-r of progeny per female

p r h d during rime ' x ' .

'Ibe mean generation time IT' was calculated from the life table

The intrinsic rate of natural increase ( r ) uas calculated using

the follaving fomla:

Page ... 31

Ihe finite rate of incream or the m m h r of time8 the population

varld aultiply itmlf if the exponential rate is continued wss

witinatad from the 'pqulatim interest rate'('r8) as below:

r Finite rate of increase ( h = e

The time required for doubling the poprlation (t) was determined

(Ohftr, 1971) a~ f ~ l l m :

'the ex~ccted values of finite rate of increase as carputed above

were rorpsred with the abQerved values of obtained f ran relative

derrsity rrPsmrrements of the population (Indoar resting) through

different months after entinrat inq the intrinsic rate of popllation

incrgase ( r ) for the successive m t h s adopting the formula:

L n D - t n D t 2 tl

m r v + j d r . ---.. ---------- - ( t - t 1 2 1

*re 'D ' is the density of the mnth of observation, t 2

'D ' is the h i t y recorded in the previous m t h and t l

(t - t 1 is the nudmr of days between two 2 1

aborvat i w . r

(brwrved finite rate of increase ( h ) = e

The deviatidns on the absented values of ~ r m the expected

v a l m of A were also calculated.

Iha dilniniuhing paportions of females with constant daily

mortality *i& m ~ w i v e to nd,m=qtrnt ovipnnitinrrn form a qmrr~tric 2 J 4 n

progte~~lion : a, art ar , ar , ar , ......... ar . (Cuellar,

1969). 'he averbge nuntier of wipositions (S) and the average rnmber

of eggs laid per emerging female (El were estimated fran this

where a r firm tenn of the geanetric progression and r = camrxl ratio of the series.

4.3. Vectorial potential:

4.3.1. Natural infection and infectivity raters:

Infecticm status of Nwumnioides nasquitoes was determined by

diaaecting the specimens abtained fran indoor resting, hunw biting

and exit trap collections for a period of four years (1986 - 1990). mila infection rate is the percmtsge of mosquitoes harbouring any

of the &velopmamtal stages of - 8. mlayi, infectivity rate is the

ptcmtmge having infective (L3) steges aut of total nmber

d i m & .

Page ... 33

4.3.2. Susceptibility status of fbrmnioides to B. malayi parasite:

One hundred freshly merged M. - annulifera females wre fed on a 3

hvnrrn microfilaris carrier (28 microfilariae per 20 na 1 and

srnples amuq the fed o m s uere dissected on daily intervals to

&tennine the duration of the extrinsic cycles. The parasite

densities per infected mosquito uere also recorded.

4.3.3. )tUMR Blood Index (HBI):

Blood samples of fullfed specimens of - M. armulifera and

M. unifonnis collected fran resting collections uere -red in a - Mrstman f i lter paper (Numt>er - 0) and these snears were subjected to

p-crcipitin testa to determine the source of blood meal. HBI vas

cstirrinted fran the results a5 folloua, which vquld give a measure of

the host select i m .

HBI = Tha proportion of b l d meals on man.

Host efficiency:

Iha index of hosrt, efficiency was calculated for - H. annulifera

and - N. uniformis fran indoor resting collections, using a slightly

Page . . .34

*re 3 Ll/fawle was wed instead of microfilariae/fen\ale as

proposad by Ksrtarsnt aincv the development of microf ilariae to first

instar of B. - mlayi took only less than 12 hours for Mansonioides.

4.3.5. Indices of trammission:

Various transmission indices have been prapased for filariasis by

different authors. Ihe m u a l trananiesion index (Baye and Gurian,

1%0), the risk of infection index (De Meillon -- et al. 1967) and the

a n w l transmission potential (WHO, 1984) were calculated for

Annual tranmnission index (AT11 = Annual biting rate X proportion infective X worm burden (mean nurt>er of infective larvae per infective nrosquito.

Risk of infectim index (RII) = Biting rate per man per hour X prooort ion parous X proportion infective to parous.

Annual trananimion potential (ATP) is a modified annual

trarum~issiorr index calculated by sunninq up of the values of mnthly

tranmninsion indices.