Hemagglutinins in mosquitoes and their role in the immune response to Brugia malayi (Filarioidea: Nematoda) larvae

Cvmp. Biochem. Physd. Vol. IleA, No. 4, pp. 1321-1326. Copyrighr 0 1997 Elscwer Scwncr Inc. All rights reserved.

1997

ISSN O’JOO-962Y/97/$17.00 IYI Sc?311@-962‘~(‘)7~L1024h-h

ELSEVIER

Hemagglutinins in Mosquitoes and Their Role in the Immune Response to Brugia makyi (Filarioidea : Nematoda) Larvae _7.K. Nayar and].

W. Knight

FWRIDA MEDICALENTOMOLOGY LABORATORY, IFAS, UNIVERSITYOF FLORIDA,200 YTH STREET.S. E., VERO BEACH,FL 12962, U.S.A.

ABSTRACT.

Hemagglutinins

to Brugia malayi

were determined

(Filarioidea:Nematoda).

High

and in the body fluid of a completely

refractory

Anophefes

ydrimculatzcs;

(Black-eye,

Liverpool

strain),

a susceptible

completely

refractory

species

of mosquiroes,

which

blood

coagulated

hemocoel.

It ib suggested

exposed

stimulated

ot several

response

ofB. ma&

movemenr

ahnormal

glands.

WORDS.

sheaths

anJ

extract hpecies.

ai Aedes mgypt~

A&.\

ofAe.

glands

from

three

alhopicttl,

in

tclenio-

the miJgut

to the

ttl rhe glycoconlugareh

developing

ahnormal

larvae

(I_,)

formed

capsule\

that

suhacquentlg

sheaths

and sheathed

micn~filariac

of microtilarial

L, in the rhor;kcic

The reds

susceptibility/refractoriness

COMPBIOCHEMPHYSIOL118A;4:

FroPhenoloxiclases).

micrdilariae

glycoconjugates

in melanlzarion

developing

and

in the hemoc~~el hod

on the microfilarial

and resulteJ

tluiJ

in rhe salivary

of sheatheJ

present

glad

refractory

was not CounJ in the clthcr

nppalpus,

titers of hemagglutinina

hemagglutinin-hound

in the salivary

of vector

C&x

and retractor\

in the salivary

minimal cncapaularion

muscles. Only

microfilariae was ohscrved III the hemoc~~el of the other four specie> of muaqulfL)es

hemagglutinins factors

High

are susceprihle

in the body

nctivLty

quinqt&cintu.\,

rapid

present

These

anJ intracellularly

and melanization

KEY

cells.

the immune

lacked

facilitatect

were also present

Hemagglutinating

C&X

that

were found

taeniorhynchus, anJ in partially

Aedrs

of hemagglutinins

after ingestion.

moieties

muscle

in the hemoccd

species,

species.

of mosquitoes

of hemagglutinins

that high titers of hemagglutintns

carbohydrate

in rhe thoracic

that

rapdly

and An. ydrimacuhtus

rhynchus with

hut low levels

in six species titers

Bncgia ma&,

encapsulation,

filarial

suggest

through

132 1-l 326, parasite>,

that

immune 1997.

tissue specific reactions 0

1997

hem&utinina.

hemagglutinin,

(encapulation, Elsevicr immune


;Ictivatcon

Sctence

ot

Inc.

response,

melani:‘lrion.

masquitoes

INTRODUCTION Filarial

nematodes

Sheathed

use arthropods

microfilariae

late in the blood by feeding through

the foregut

vertebrate

to the

midgut.

most microfilariae

midgut

while

Fnlm

hosts

They are carried

of mosquitoes, hemocoel.

as intermediate

hosts.

of Brugia malayi and B. pahangi circu-

of their

mosquitoes.

and/or

penetrating

the hemocoel

and are ingested

with the bloodmeal

In susceptible

cast their the

sheaths

midgut

species in the

wall to the

they migrate

to the thorax

where they invade the thoracic muscle cells and develop intracellularly to the infective third (L>) stage larvae. Infective larvae throughout

mouth

the

leave the muscle the body cavities

ence for the mouth

cells and distribute of a mosquito

parts. Transmission

themselves

with

of these

a prefer-

larvae from

Address rcpmr requ~sr~ to: J. K. Nayar, Florida Me&cd Entomology Lahoratory, IFAS, Ilnivcrsq~ <>fFlorida, 200 9th Street, S. E., Vera Beach, FL 32962. U.S.A.; Tel. (561)778-7200; Fax (561)778X05; E-mail: Ikn@ ic,,n.\,en,.ufl.c‘lu. Recewcd 23 I)ecemher 1996; revised 29 April 1997; accepted 26 May 1997.

parts to the vertebrate

tive mosquitoes However,

take another

not all species

host occurs

bloodmeal of mosquitoes

of refractoriness

to these

infec-

are susceptible

B. malayi and B. pahan,@. Most species grees

when

(5). exhibit

filariae.

Several

varying

to de-

refractory

mechanisms are shown to be present in refractory species: (a) Immediately after ingestion of microtilariae by some species of mc>squitoes, pharyngeal

microfilariae

armatures

within

encounter the head

the cibarial

capsule

and

of the mos-

quito. The cibarial armature is a set c~f teeth that project into the lumen of the ciharium, while the pharyngeal armature is a set of sharp lumen

spines

of the pharyngeal

Aanically

abrade

that pump.

the sheaths

project Both

into the posterior armatures

and cuticle

may me-

of microfilariac

as

they pass through the foregut, but the c&aria1 armature is the most lethal (5). (b) Shortly after feeding, the blood in the midgut of some species of mosquitoes forms a clot. The speed of clotting varies among specie>. Microt;lariae may become trapped in blood clots when blood coagulates quickly midgut.

and are prevented In those species

from penetrating (>f mosquinlch

the wall of the in which blood

1322

J. K. Nayar

slowly coagulates

in the midgut,

some or most of microfila-

riae leave the midgut within 3 to 24 hr (5). (c) Even though microfilariae may evade all of the refractory mechanisms that operate hemocoel

in the midgut

muscles,

invade

their development

tibility

to filarial infection

trolled

by simple

dominant

is an inherited

sex-linked

species

grate successfully are other

susceptible

Suscep-

characteristic

conbeing

( 12).

either

rapidly

in which

microfilariae

mi-

or slowly to the hemocoel,

that control

and refractory

their future fate. In both

mosquito

species,

most of the mi-

crofilariae migrate from the midgut to the hemocoel, invade thoracic muscle cells and start development intracellularly as first stage

(L, larvae).

In the susceptible

species,

aegypti (Black-eye,

Liverpool

strain),

ment

L, within

11 to 13 days while

and become

generate

(1). In partially

rimaculatus,

some

hemocoel while

and

most

muscle

refractory

of the

they

of the

develop

microfilariae

to L1 within

species,

others

Anopheles

are trapped

encapsulated

cells start development

Aedes

most L1 start develop-

microfilariae

become

that

and

invade

dequad-

the

thoracic

to L, and a portion

lated and melanized

both

to the hemocoel

in the hemocoel

of them and

and melanization mosquito cent

to abnormally

studies

glands

from

where hemagglutinins present

refractory and encapsulation response

Brugia larvae.

that hemagglutinins

of An. quadrimaculatus

of microfilariae

jugates

by an immune

developing

(17) suggested

the salivary tion

is caused

are encapsu-

and in the tho-

racic muscle cells (11,25). In both partially completely refractory species of mosquitoes,

the

midgut

of the Our re-

present

facilitate to the

from the hemocoel

on the microfilarial

in

migra-

This

in the hemocoel study

presents

hemagglutinins response

that

microfilariae

and of L, in the thoracic

further

in Florida results

evidence mosquitoes

in encapsulation

in the hemocoel

that

l-2

2-8

64-512

256-512

64-128

128 16-32

“Hemagglutinat~on titers rxpresscd as the reciprocal (l/n) of the In occurs. t20 pr of SG of all species except Ae. tuentorhynchus were extracted in 35 ~1 of LI’BS and 5 ,ul of the extract was used for making each wnes uf dilutions to derrrmme HA activity. In Ae. r~ienitrrh$u only 10 pr of SC; were used. :l:The body fluld was extracted from 150 females of each apturs and only 5 ~1 of the body fluid was used for making each series of dilutions TCIdetermine HA activitv.

dium

pentobarbital

(Meriones

anesthetized

ungiculatus)

microfilariae/@ each species

blood).

Groups under

of 50 blood-fed

12L: 12D cycle.

from each

group

240 hr after

from each

and maintained

Subsequently,

infective

females

of

ingested species

10 blood-fed

were

females

at 4, 24, and

as described

microfilariae

was

at 25 2 1°C

individually

bloodmeal

(15) and the fate of ingested

(25 to 30

in a drop of saline

of microfilariae

were dissected

the

blood-fed

dissected

females

soon after feeding

jirds

parasitemias

Ten individual

solution, pH 6.8 and number determined. separated

B. ma&-infected

with moderate

were immediately

Preparation

muscles.

the presence

induces

1

-

earlier

was determined.

Determination of the Presence of Hemagglutinins

and abnormally

developing larvae and result in encapsulation and melanization of B. ma& microfilarial sheaths and sheathed microfilariae

quinquefhsciatus nigripulpzcs albopictus taeniorhynchus quadrimacdatus aqypi (Black-eye)

hemocoel,

bind to glycocon-

sheaths

Cx. Cx Ae Ae. An. Ae.

Body fluid*

in the

11 to 13 days, some degenerate,

from the midgut

Salivary glands?

Mosquito species

melanized,

others become encapsulated and melanized (18,19). In completely refractory species, Armigeres subalbatus, microfilariae that migrate

Hemagglutination titers in

into the

alleles with refractoriness

of mosquitoes

factors

migrate

TABLE 1. Hemagglutinationtiters*in the salivaryglands extract and in the body fluid of females of six species of mosquitoes against Human A+ erytbrocytes

the cells of the thoracic

to L, is not guaranteed.

to susceptibility

In those there

and successfully

and subsequently

and J. W. Knight

of

an immune

and melanization

and of L, in the thoracic

of mus-

of red blood

(SG) extract,

collection

hemagglutination for hemagglutinins

cells

salivary

gland

blood was obtained Vero

Beach,

and

assay and carbohydrate were described earlier

blood was used for these studies against

(RBC)

of body fluid (mostly

extract

salivary

gland

hemolymph),

inhibition tests (17). Human A+

as it gave the highest

titers

and the body tluid (17). The

from the Indian

River Blood Bank Inc.,

Florida.

cle cells.

MATERIALS AND METHODS Mosquitoes Used and Infection with Filariae Six species of mosquitoes maintained in our laboratory were used for theses studies: Cukx nigripalpus, Cx. quinquefasciatus, Aedes taeniorhynchus, and Ae. albopictus. The selected refractory strain of An. quadrimaculatus tible Ae. aegypti (Black-eye, Liverpool

(19) and the suscepstrain) were used as

controls. Groups of 3- to 5-day-old (1100) of each species were blood-fed

sugar-fed females to repletion on so-

RESULTS Hemagglutination (HA) in the Salivary CJmds (Scj) and the Body Fluid Higher titers of HA were found in the SG extract of Ae. taeniorhynchus and An. quadrimacukztus against RBCs from Human A+, but none were found in the SG extract of the other four species (Table 1). Substantially higher titers of HA were found in the body fluid of Ae. taeniorhynchus (1: 256-512) and An. quudrimaculatus other species of mosquitoes (Table

(1: 128) than 1).

in four

Hemagglutinins

in Mosquitoes

1323

2. Distribution of ingested microfdariae of Brugia malayifrom the midgut to the hemocoel of six species of mosquitoes 24 hr after the infective bloodmeal, encapsulation and melanization of microfilariae in the hemocoel and L, in the thoracic muscles and development of Lz and L3 in thoracic muscles 240 hr after the infective bloodmeal

TABLE

240 hr after ingestion* Encapsulated and melanized mfs, smf, and L, in

24 hr after ingestion*

Mosquito

mf ingested X + SE

species

Cx. quinfpefasciatus

30.0 ? 8.8

Cx. nigripalp.a

28.7 1- 5.8

Ar alhojictus

30.6 2 5.9

Ar. tueniorhynchus

26.7 5 3.4

An. quadrimaculatus

50.2 ?I 9.6

Ae. aempti (Black-eye)

24.7 ? 3.8

mfin midgut X + SE

mmfs in hemocoel and L1 in thorax X rt SE

(%)

W)

27.5 ? 3.1 (98.9) 24.2 ? 2.2 (93.0) 26.6 t 2.9 (95.0) 8.1 i 1.9 (33.8) 2.1 i- 0.4 (4.1) 10.5 t 0.6 (43.4)

*Thirty females/group/time were dissected m three experiments. mfs = Microfilarial sheaths; mf = microfilariae; mmfs = melanized

of migration

crofilariae of B. dayi

of sheathed

opment

Thoracic muscles X k SE

0.3 + 0.1

0.0

0.0

0.0

0.0

0.0

0.5 ? 0.3

0.0

14.6 -e 1.7

3.3 i

0.0

18.0 i- 2.2

4.0 -e 1.4

5.6 -c 0.9

1.7 + 0.4

0.8 -e 0.3

18.5 + 2.3

3.1 + 0.6

I.2

can also be divided into the same two groups as

above. In the first group of mosquitoes (CX. quinquefusciatus,

and exsheathed

mi-

in the six species of mosquitoes from

the midgut to the hemocoel24

L, X f SE

microtilariar; smf = bheathed microfilariae

Migration, Encapsulation and Melanization, and Dewelopment of Ingested Microjilariae Comparison

Thoracic muscles

Hemocoel mfs + smf X + SE

0.5 ? 0.2 (1.1) 1.9 ? 0.4 (7.0) 1.4 2 0.4 (5.0) 15.9 5 0.9 (66.2) 45.9 + 3.2 (95.9) 13.5 + 1.2 (56.6)

Development of L2 and L, in

hr after the infective blood-

Cx. nigripalpus, and Ae. albopictus), the few microfilariae that migrated into the hemocoel

became encapsulated

and

melanized, and none of them reached the thoracic muscles

two groups. In the first group of mosquitoes (Cx. quinquefas-

(Table 2). In the second group of mosquitoes, there were distinct differences among the three species in the numbers

ciatus, Cx. nig-ripalpus, and Ae. albopictus),

of microfilarial

meal showed that the migration

rates can be divided into only a few mi-

crofilariae,

1.1% to 7.0%, migrated from the midgut to the

hemocoel,

and in the second group of mosquitoes (Ae. taeni-

orhynchus, Ae. aegypti, and An. quadrimaculatus), 56.6% 95.9% of the microfilariae hemocoel infective

(Table

to

migrated from the midgut to the

2). Similar comparison

at 4 hr after the

bloodmeal showed that only ~1%

of the microfi-

lariae migrated in the first group of mosquitoes, second group of mosquitoes

migration

but in the

rates were variable.

sheaths and sheathed

that were encapsulated

microfilariae,

microfilariae

that entered the hemocoel

some sheathed

microfilariae

sues and both became encapsulated sheathed

microtilariae

that

cells and started development

shed their sheaths,

got entangled

in different tis-

and melanized; the ex-

invaded

the thoracic

of sheathed

(Table

had

muscle

also became encapsulated and

melanized as L, with no further development

50% and 79%, respectively,

and

in thoracic muscles, and the numbers developing to L? and L, (Table 2). In Ae. meniorhynchus, most of the sheathed

In Ae. taeniorhynchus and An. quadrimaculatus the majority microfilariae,

and L,

and melanized in the hemocoel

to Lz or Li

2). In An. quadn’maculatus, a similar situation

oc-

already migrated from the midgut to the hemocoel compared with only 10% in Ae. uegypti within the first 4 hr after

curred except that a few L, developed to L1 and L1 (Table

the infective bloodmeal

and some mi-

migrate slowly from the midgut to the hemocoel, very few microfilarial sheaths and sheathed microfilariae became en-

crofilariae that failed to exsheath in the hemocoel became encapsulated and melanized in both An. qu&imucuIatzls

capsulated and melanized as L, in the thoracic muscles, with most of the L, that invaded the thoracic muscle cells devel-

and Ae. taeniorhynchus, but only a few did in Ae. aegypti. The exsheathed microfilariae invaded the thoracic muscles

oped to Lz and L3 (Table

substantial

(data not presented in Table 2). A

number of microfilarial

sheaths

and started development. A similar comparison of encapsulation

and melanization

of L, and development to LZ and L3 of B. ma& in thoracic muscles of six species of mosquitoes showed that the devel-

2). In Ae. aqypti,

Carbohydrate

where mostly exsheathed

microfilariae

2).

Inhibition

Since higher titers of HA in the SG and in the body fluid were found in Ae. taeniorhynchus and An. quadrimaculatus,

J. K. Nayar and J. W. Knight

1324

3. Degree of inhibition of agglutination of Human A+ erythrocytes by salivary glands extract and body fluid of Aedes taeniorhynchus (AT) and the refractory strain of Anopheles quadrimaculatus( AQ) * to B. malayi by 300 mM or less of various

TABLE

carbohydrates. Degree of inhibition, compared to positive controls, scored as: - = negative; f = reduction in end-point titer by one well dilution; 1 = two well dilutions; 2, 3, 4,....: two, three, four,,... well dilutions, respectively Body fluid

Salivary glands AT

Carbohydrates

DLArabinose? L( - )Fucose D(+)X$OSe

D( + )Glucose

D( + )-Mannose Maltose D( +)Trehalose Stachyose D( f )Melezitose D-Gluconic acid n-Glucuronic acid wGalacturonic acid D( +)Galactosamine HCI D( +)Glucosamine HCl n-Mannosamine HCl N-Acetyl-D-galactosamine N-Acetyl-D-glucnsamine N-Acetylmuramic acid N-Acetylneuraminic acidS Methyl &D-glucoside Methyl Pbglucoside Methyl adn-galactoside Methyl au-galactoside p-Nitrophenyl cx-D-Galactoside$ ~Nitrophenyl a”o-Galactoside4

carbohydrate

inhibition

body fluid against two species.

of HA activity Human

_

_ _ _ 1 6 _ _

1 1

3 _

+

1

L

1

2 _

2 ? 2 5 ? 2 2 t-

2-3 _ _ 1

2 5 ? +_

4-5 _ _

3 i

_

2

_

1

1 2

_

1

_

1

of the SG and the tested

in these

for inhibition

of An. ql*adrimacutat~ ine and partially carbohydrates

was largely inhibited

inhibited

(Table

in varying

by mannosam-

degrees

by 14 other

3).

and the body fluid of fe-

and An. quadrimaculatus

by N-acetylneuraminic

i +

_

A+ RBC were compared

showed

inhibition

tueniorhynchus than in An. quudrimaculatzls. ity in the SG extract of Ae. taeniorhynchus inhibited

_ _ _

buffered saline (DPBS).

of HA activity

A+ RBC, fewer of them

t

2 -e + 1 1 _ _

_

of the SG extract

-

+

l-2 _

males of Ae. tueniorhynchus

+

1

_ 1 2-3

Of the 36 carbohydrates

_

i

1 1 _

1 _

Human

AQ

1

_

*Data from Nayar and Knight (1997). t651) mM solution in Dulbecco’s phosphate $80 mM solutwn m DPBS. $25 mM solution in DPBS.

AT

AQ

against in Ae.

The HA activwas completely

acid and partially

inhib-

DISCUSSION These and earlier results and An. quadrimaculatus salivary

gland extract

( 17) show that Ae. tmniorhynchus have high titers of HA in their

and in the body fluid from the hemo-

ited by arabinose, fucose, xylose, maltose, melezitose, gluconic acid, glucuronic acid, and N-acetylmuramic acid (Ta-

coel. The migration

ble 3). On

crofilariae from the midgut into the hemocoel during the first 4 hr after they are ingested. On the other hand, in Ae.

the

other

hand,

the

HA

activity

in the

SG

high titers of HA in the salivary glands allow of the major portion of ingested sheathed mi-

extract of An. qdti’macuhtus was almost completely inhibited by N-acetyl glucosamine and partially inhibited in varying degrees by 21 other carbohydrates (Table 3).

aegypti sheathed and exsheathed microfilariae migrate slowly over a 24-hr period from the midgut into the hemo-

The HA activity of the body fluid of Ae. taeniorhynchus was largely inhibited by N-acetylneuraminic acid and partially inhibited by gluconic acid, glucuronic acid, galacturonit acid, galactosamine, glucosamine and N-acetylmuramic acid. On the other hand, the HA activity of the body fluid

coel. However, we could not demonstrate the presence of HA in the salivary gland extract in Ae. aegypti (Black-eye, Liverpool strain), even though hemagglutinins have heen demonstrated in the SG extract of other strains of this speties (7). Our results confirm the presence of hemagglutinins

Hemagglutmms

in Mosquitoes

1325

in the body fluid of the Liverpool In the other

three species,

pus, and Ae. albopictus, the midgut though

and in these

the presence

a very small amount

body fluid. These

of Ae. uegypti (23). Cx. nigripal-

only a few microfilariae

to the hemocoel,

not demonstrate

strain

Cx. quinquefasciatus,

species

migrate

species

from

we could

of HA in the SG extract of activity

even

was detected

are refractory

in the

to infection

of B.

also bind to the glycoconjugates moieties sheathed

their melanization.

the midgut

the

midgut

to the hemocoel,

species

(5). These

y&rimaculntta,

with

ingested

the

midguts

which

allows

midgut

into

freer

the

SC; extract

shown

that

from

of the

jirds from

interpretation

quadrimaculatus the

present

facilitate

midgut

the

to the

in

move-

rapid migration

the hemocoel tus, most

of microfilariae

sheathed

microfilariae they enter

from the midgut

to

and An. quadrimacula-

of Ae. taeniorhynchus

still retain

ial sheaths

when

Ae. aqypti

the lack of HA in the salivary

their microtilar-

the hemocoel.

In contrast, gland

in

clots that probably

slows down

riae in the midgut,

with the result that most of the microfi-

lariae

exsheath

exsheathed

in the midgut.

of plant

our earlier

moieties

microtilariae

and to intracellularly

on microfilarial

demonstrated

to characterize

migrate

slowly

conclusions

(15).

with exposed

sheaths

and sheathed

developing

earlier

the carbohydrate

20). It is suggested

of the

to glycoconjugates

carbohydrate vae has been

most

microfilariae

reaffirming lectins

of microfila-

Subsequently,

and a few sheathed

into the hem<)coel, Binding

the movement

abnormal

lar-

in the midgut

moieties

that hemagglutinins

(Z-4,6,10,16,19, present

bind to the glycoconjugates

moieties

present

These hemagglutinin-bound to the hrmcjcoel, where

in the sali-

mation

and sheathed

of capsules

dase system,

which

that,

prophenoloxidase quadrimurulutus

with exposed

microfilarial

of hemagglutinins microfilariae

in turn,

melanizes

few hour5 after reaching

the

car-

sheaths.

sheathed microfilariae migrate most of them exsheath but some

of them do not. The binding larial sheaths

on

ex-

cells, with

both

Earlier,

capsules.

He demonstrated

ies where

seem to attach

Our results

in the body tluid and

speciticities

hemagglutinins

that agglutinate

man A+ RBC are present

in the SG extract

fluid of sugar-fed

of An.

females These

nonspecific

ent in other Finally,

mosquitoes

our results

and insects

of Florida

of sheathed

midgut

to the hemocoel,

and melanization

mosquitoes, and

of microtilariae

oc-

to those pres-

relationship

in the salivary

and exsheathed

and Ae.

naturally

(8,9,13,17,22-24)

show a distinct

gration

in the thoracic

and are similar

of hemagglutinins

in the hemocoel

are

Hu-

and the hody

qwdrimaculatzcs

hemagglutinins

molecules

and f<>rm in vitro stud-

titers.

also show that heterogenous carbohydrate

that

to the sur-

in the hemvcoel

microfilariae

and in

(2 1) suggested

this by conducting

he incubated

of mos-

the immune

in the hemocoel

Ogura

a loss of hemagglutinin

with varying

trigger

between glands

and

with the rapid mimicrofilariae

the e\Fentual

from the

encapsulation

in the hemocoel

and L,

muscles.

.Iird, infected with Brugia

malayi used in this study were ~ov~d~d by u NIAID Supply Contract (Al-026421 I_‘. S. Japan Cooperative Medicul Science Program. Unioersity of Florida, Institute of Food and An_ ricultural Sciences Experimental S&ions Journal Series-No. K-05478.

in An. qz~&macularus

vary glands of Ar. taeniorhynchus and An. quadrimacufatus when mixed with the ingested blood containing B. malayi bohydrate

larvae

that

of Ar. subalbatus

the presence

the blood

muscle

in these species

molecules

muscles.

hemagglutinins

curring

(15,17). With

Hemagglutinins

to B. ma&

thoracic

taeniorhynchus.

hemocoel

on trapped

and on intracellularly

L, in the thoracic

act as immune

response

showed

confirms

hemagglutinins

from

in

erythrocytes

of microfilariae This

that

of An.

SG mix

B. malayi-infected

movement

hemocoel.

of micrafilariae

in the

microfilariae

face of B. makxyi microfilariae

in Ae. taeniorhynchtrs

agglutination

conclusions

from the

in some other

hemagglutinins

blood

the

earlier

ment

as has been

causing

our

of microfilariae

results suggest

and An. their

migration

abnormal

carbohydrate

sheaths,

the result that they become encapsulated. These capsules in turn trigger the prophenoloxidase system that results in quitoes

inhibit

with exposed

on the microfilarial

and sheathed

developing

malayi and they may either possess cibarial and pharyngeal armatures in the head capsule or have clotting factors in that

present

trigger

in the for-

the prophenoloxi-

the capsule

the hemocoel.

on microfi-

results within

The presence

system was demonstrated and Ae. argypti (14). The

the first of the

earlier in An. hemagglutinins

present in the hemocoel also encapsulate the microfilarial sheaths nncl sheathed microtilariae in the hemocoel that later become melanized. The hemagglutinins present in the body fluid from the hemocoel of AC. tneniorhynchus and An. quadrimacufatus

References 1. Beckett, E.B.; Mac&&J, W.W. The hurvival nnd rfcvelopmrnt of subperiodic Brugin makqi and B. p&n~ in ‘1 xlecteJ strain of Aedes ueg?lpti.Trans. Royal Sot. Tnlp. Hyg. 65~139~ 346;1971. 2. Bra&y, T.J.; Nayar, J.K.; Ch’k’l’: I I Im, M.L.; Knight, J.W.; Mikarts, L.L. Ultrastructural localization of lectin bindmg sites cjn larvae nf Brugia ma& (Nematoda: Fllarioidea) clevelk)ping in Anophe/es qwdrimac&tt*c. In: Borovsky. D.; Spielman. A. (eds). Third Symposium Proceedings of Host Regulated Developmental Mechanisms m Vector Arthropuds. Vera Reach, FL: Florida Medical Entomology Laboratory, University of Florida; 1993:209-2 17. 3. Chikilian, M.L.; Bradley, T.J.; Nayar, J.K.; Knight, J.W. Ultrastructural comparison of extracellular anil intracellular encapsubion of Brugia malayi in Anophelrs qwdrimnculatu~. J. l’arasitol. 8@:133-140;1994. M.L.; Bra&y, T.J.; Naynr, J.K.; Cash-Clark, C.E.; 4. Chikilian, Knight, J.W. Ultrastructure of the intracellular melanization ot Brzcgia maI+ (Buckley) (Nematoda: Fllarioiciea) in the thorscic muscles of Anopheles quudrimaculntus (Say) (Diptera : Culicidae). Int. J. Morphol. Emhryol. 24:X3-92;1995. 5. Denham, D.A.; McGreevy, P.R. Rruglan tilnnasis: Epi&mi~~-

1326

6. 7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

logical and experimental studies. Adv. Parasit. 15:243-309; 1977. Devaney, E. Lectin binding characteristics of Brugia pahangi microtilariae. Tropen. Med. Parasit. 36:25-28;1985. Hudson, A. Some functions of the salivary glands of mosquitoes and other blood-feeding insects. Can. J. Zool. 42:113120;1964. Ingram, G.A.; Molyneux, D.H. Insect lectins: Role in parasite-vector interactions. In: Kilpatrick, D.C.; Van Driessche, E.; Bog-Hansen, T.C. (eds). Lectin Reviews. St. Louis, MO: Sigma Chemical Company; 1991:103-127. Ingram, G.A.; Molyneux, D.H. Comparative study of hemagglutination activity in the hemolymph of three tsetse fly Glossinu species. Comp. Biochem. Physiol. 106B:563-573;1993. Kaushal, N.A.; Simpson, A.J.G.; Hussain, R.; Ottesen, E.A. Brugia malayi: Stage-specific expression of carbohydrate containing N-acetyl-D-glucosamine on the sheathed surfaces of microfilariae. Exp. Parasitol. 58:182-187;1984. Kobayashi, M.; Ogura, N.; Tsuruoka, H.; Chigusa, Y.; Mishima, S. Studies on filariasis VII: Histological observation on the encapsulated Bru@ mu& larvae in the abdominal haemocoel of the mosquito, Armigeres subalbatus. Jpn. J. Sanit. Zool. 37:59-65;1986. Macdonald, W.W. Mosquito genetics in relation to tilarial infections. In: Taylor, A.E.R.; Muller, R.L. (eds). Genetics Aspects of Host Parasite Relationships. Symposium Brit. Sot. Parasit. 14:74-87;1976. Mohamed, H.A.; Ingram, G.A.; Molyneux, D.H. Carbohydrate-binding specificities of anti-erythrocyte lectins (haemagglutinins) in Anopheles gambti gut extracts and hemolymph. Med. Vet. Entomol. 6:217-224;1992. Nayar, J.K.; Bradley, T.J. Comparative study of hemolymph phenoloxidase activity in Aedes aegypti and Anopheles quadntnuculatus and its role in encapsulation of Brugio malayi microfilariae. Comp. Biochem. Physiol. 109A:929-938;1994. Nayar, J.K.; Knight, J.W. Comparison of migration and encapsulation of Brugia m&i microfilariae from the midgut to the hemocoel between AnopheLs qtuufrimaculatus and Aedes aegyrpti. J. Invertebr. Pathol. 65:295-299;1995. Nayar, J.K.; Knight, J.W. Cytochemical localization of lectinbinding to intracellularly melanized first stage larvae of Brugin dayi (Buckley) (Nematoda:Filarioidea) in Anopheles q&Ii’maculatus (Say) (Diptera : Culicidae). Int. J. Insect Morphol. Embryol. 25:331-339;1996.

J. K. Nayar and J. W. Knight

17. Nayar, J.K.; Knight, J.W. Hemagglutinins in Anopheles quadrimaculatus, strains susceptible and refractory to Brugia dayi, and their role in the immune response to filarial parasites. Comp. Biochem. Physiol. 116B:109-117;1997. 18. Nayar J.K.; Knight J.W.; Vickery A.C. Intracellular melanization in the mosquito Anopheles quadrimacufutus (Diptera: Culicidae) against the filarial nematodes, Brugia spp. (Nematoda: Filarioidea). J. Med. Entomol. 26:159-166;1989. 19. Nayar, J.K.; Bradley, T.J.; Knight, J.W.; Mikarts, L.L. Development of Brugia malayi (Nematoda: Filarioidea) larvae in susceptible and refractory strains of Anophefes quadrimacuktrus characterized by lectin binding. In: Borovsky, D.; Spielman, A. (eds). Third Symposium Proceedings of Host Regulated Developmental Mechanisms in Vector Arthropods. Vero Beach, FL: Florida Medical Entomology Laboratory, University of Florida; 1993:218-233. 20. Nayar, J.K.; Mikarts, L.; Chikilian, M.L.; Knight, J.W.; Bradley, T.J. Lectin binding to extracellularly melanized microfilariae of Brugia malayi from the hemocoel of Anopheles quad+ maculatus. J. Invertebr. Pathol. 66:277-286;1995. 21. Ogura, N. Haemagglutinating activity and melanin deposition on microfilariae of Brugia p&ngi and B. m&i in the mosquito, Armigeres subalbatw. Jpn. J. Parasitol. 35:542-549; 1986. 22. Ogura, N.; Kobayashi, M.; Yamamoto, H. Haemagglutinating activity in fluid collected from the mosquito, Arrnigeres subalbutus, by centrifugation method. Dokkyo J. Med. Sci. 12:2 17221;1985. 23. Ogura, N.; Kobayashi, M.; Yamamoto, H. Haemagglutinating activity in fluid collected from the mosquitoes, Aedes togoi and Aedes aegypti by centrifugation method. Dokkyo J. Med. Sci. 12:223-226;1985. 24. Rowley, A.F.; Ratcliff, N.A.; Leonard, C.M.; Richard, E.H.; Renwrantz, L. Humoral recognition factors in insects, with particular reference to agglutinins and the prophenoloxidase system. In: Gupta, A.P. (ed). Hemocytic and Humoral Immunity in Arthropods. New York: John Wiley & Sons; 1986: 381-406. 25. Yamamoto, H.; Kobayashi, M.; Ogura, N.; Tsuruoka, H.; Chigusa, Y. Studies on filariasis VI: The encapsulation of Brugia malayi and B. pafumgi larvae in the mosquito, Armigeres subalbatus. Jpn. J. Sanit. Zool. 36:1-6;1985.