histamine in culexthe

The Japan Society of Medical Entomology and Zoology

NII-Electronic Library Service

The JapanSociety of Medical Entomology and Zoology

[Jpn. J. Sanit. Zool. Vol. 36 No. 4 p. 315-3261985]

Histamine

theand

esterases in

mosquito, Culexthe salivary gland of

pipiens paltens

Yasuhiro NAKAyAMA,' Fumihiko KAwAMoTo,' Chiharu SuTo,'

Terumi NAKAJiMA,*""" Tadashi YAsuHARA," Hisashi FuJioKA'

and Nobuo KuMADA*

*Department

of Medicat Zeotegy, Nagova University School of Medicine,

65 Tsurumai-cho, Showa-ku, Nagoya 466, Japan**Division

of Molecular Bielegy, lnstitute foT Medical and Dental Engineering, Tokyo Medicat

and Dental University, 2-3-10, Kanda-Surugadai, Chiyoda-ku. Tokyo 101, JaPan

***Fatulty ef Pharmaceutical Sciences, University of Tokye,

7-3-1 Hongo, Bunkyo-ku. Tekyo 1l3, Japan

(Received: April 8, 1985)

Key words: mosquitoes, skin reaction,salivarygland,histamine,esterases.

Abstract: In an attempt to elueidate the mechanism of skin-reactions to

mosquito bites, components in the salivary gland of mosquitoes were studied. Crude

extract and Iower molecular weight fraction (mol. wt. <6,OOO) of the salivary glandextract of Culex PiPiens paltens showed extravasation activity in the skin of guinea

pigs net sensitized previously. But higher molecular weight fraction (mol. wt. >6,OOO)

of the extract did not have the extravasation activity. By the o-phthalaldehyde

staining method, histamine was detected in the whole $alivary glands of Cx. pipiensPallens soon after their emergence, and the reaction intensity increased when they

were mature; in Aedes togoi, the proximal portion of the lateral lobe of the salivary

gland reacted strongly. By the azo dye staining method, using a- and B-naphthylacetate, strong esterase activity was found in the whole salivary gland of Cx. pipienspallenss in Ae. togoi and Ae. alboPictus, only the proximal portions of the salivary

glands reacted to some extent. In vitro tests with the salivary gland extract of Cx.

pipiens pallens did not exhibit hydrolytic activity on TAME, BTEE and ATEE,

and neither anticoagulating activity nor hemolytic activity was observed. Histamine,

putrescine, spermine, and spermidine, but not serotonin, were detected in the

salivary gland extract by ion-exchange high performance liquid chromatography.

Histamine contained in the female body was mainly found in the salivary gland and

head,

*tliLkeege, Mpt1th, utee=F#, rkma fi, meM

deft: GaM ± \M\zzveijtm\.a$ (+466 k dismwa$nxrefiptI 6s)** igdeEra, fifi ec: mmll-Katwfl)k\eemeeu315

***Meent

(+IOI10)MMr"Jit\rs\ms

1>

M m- as =T: ft MX mp M wa pt S

(+113 MA-aslsc"X*as

2-3-

7-3-




316

INTRODucTION

AIthough the mosquito bites provokeadverse skin reactions in man and animals,

chemical nature of the mosquito saliva isnot fully understood. These skin reactions

may be classified into immediate, delayed,or a combination of these types. In the

immediate reaction, wheal, erythema, and

pruritus may develop within a few minutes

after the bite and usually subside in a short

time. The delayed reaction appears within

a couple of days after the bite and is char-

acterized by papular lesions, often accom-

panying edema and intense pruritus. Thesesymptoms may persist for several days.Rarely, mesquito bites cause severe systemic

reactions or even death in the highly sensi-

tive individuals (Takahashi et al. 1973;

Suzuki et al., 1976s Yasuda and Hirano,1980; Urisu et al. 1981).

Various investigators reported that mam-

mals, including man, become sensitized bymosquito bites (Mellanby, 1946; Gordonand Crewe, 1948; McKiel, 1959; Allen and

West, 1966; Gillet, l971). There are, how-ever, conflicting views as to whether any

toxic substance is involved in the rnosquito

saliva. Some investigators (Gordon and

Crewe, 1948; Rockwell and Johnson, 1952)

observed skin reactions in experimental ani-

mals not previously sensitized and in humanbabies and considered that the skin reaction

is due to some unknown toxic substances.

Eckert et al. (1950, 1951) applied Code'smethod of histamine extractien to the whole

bedy or body parts ef Culex PiPiens. Theseextracts caused contraction of guinea pigintestine, which was inhibited by histaminase

treatment But, McKiel (1959) found no

skin reaction to the bite of Aedes aegyPti

or to injectien of its extract in experimentalanimals which had not previously been ex-

posed to the mosquitoes. He considered that

there was neither histamine nor histamine-like compound in the mosquito saliva studied.

Previous attempts to purlfy or identify

specific component(s) responsible for the

mesquito bite reactions were carried out

using crude extracts of the whole body or

bedy parts that contained heterogeneous

Jpn. J. Sanit. ZcK)1.

constitutents (Eckert et al., 1950, 1951;McKiel and Clunie, 1960; Yasuda and Hi-rano, 1980), Because majority of these

constituents were considered to be not con-

cerned with the bite reaction (Feingold et

al., 1968), studies using less heterogeneousextracts collected directly from the salivary

glands have been required,

While maintaining mosquitoes in the labo-ratory, we frequentiy observed erythema re-

actions on the ears of rnice when they were

exposed for the first time to Cx. pipiensPaltens. This led us to a series of studies

on the salivary gland components, includingtheir roles in skin reactions, functions inmosquito physiology, and effects on trans-

mission of pathogens. Herein, we report the

presence of histamine and esterases in the

salivary glands of mosquitoes demonstratedby histochemical and chromatographic

methods.

MATERIALs AND METHODS

Experimental animals

Original colony of Culex piPiens paltens(Coquillett) and those of Aedes alboPictus

(Skuse) and Aedes togoi (Theobald) were

kindly provided by Fumakilla Co. and Prof.T. Oda of Nagasaki University, respectively.

They were maintained in our laboratory forsome generations before use. The larvaewere fed on mouse food (Clea Japan Co.).The adults were maintained at an approxi-

mate temperature Df 250C and 70% relative

humidity and fed with 5% sucrose solution.

Hartley strain guinea pigs reared with

rabbit food (Clea Japan Co.) were used

when about six weeks of age. They hadnever been exposed to mosquitoes before

use.

Collection of salivar7 glands from mosguitoes Female adults were anesthetized by chillingin a freezer for about ten minutes, then

dissected in Clarke's solution (distilled water

200ml, NaCl 1.3g, KCI O.028g, CaC12

O,024 g, NaHC03 O.02 g, Na2HP04 O.O02 g)or saline under a binocular stereomicroscopeusing micro-tweezers and a 27 gauge injec-

tion needle. After dissection, salivary glandswere promptly subjected to histochemical




Vol. 36 No.4 1985

staining, For biQchemical tests, the glandswere transferred into a small amount of

saline or CIarke's solution and stored in a

-300C deep freezer until use.

Ex'tracticn of comPonents from salivary

.alands and bod), parts

Stored salivary gla,ncls, midgut, crop, and

head and thorax were homogenized by ultra-sonication and incubated at 40C overnight,

then homogenates were centrifuged at about

400 g; and the supernatants, or crude ex-

tra,cts, were used for the tests describedbelow. In some experiments, extract from

seme five hundred salivary glands was frac-tionated ",ith a sing.le hollow fiber concen-

trator (SHFC, Biomed Instrument Co.) to

obtain hi.crher and lower molecular weight

fractions (HMF;, mol. wt. >6,OOO, LMF;mol. wt, <6,OOO). These procedures were

performed at 4"C or in an ice bath, Foranalysis by high performance liquid chro-

rnatography (HPLC), the stored salivary

gland, the whole body, head, thorax and

abdomen were lyophilized, and suspended

in 250 sLl of 10C)F trichloroacetic acid (TCA)and homegenized with a glass rod, then

centrifuged at about 400 g. The supernatants

were used for HPLC analvsis. i

E;t'travasation test irt guinea Pibas Hartley strain guinea pigs weighing about

300g and not sensitized previously by mos-

quitoes were injected intracutaneously ",ith

O.05 ml of crude extracts on the shorn back,The injected ainount was expressed by sali-

x.ary gland (or number of female) equiva-

lents as descrlbed in Fig, 1 and Table 3.Ervthema or other skin reactions at the in-

jec'ted sites were observed 15 minutes later,

Then, e.5ml of O,59 Evans blue in saline

was injected into the femoral vein. Thirtyminutes after the dye injection, these guineapigs were sacrificed and bled by N,enesection,

then the back skin was exfoliated with a

scalpel. I)iameter of the blue spot was

measured and eompared with that of the

saline injectcd control. A test was con-

sidered positiiJe when the diameter of bluespot exceeded 4.0min, accerding to the

c:riterion by Newsorne et al. (1969).

317

Histochemical staining ot salivarJ, .aland

1) Histamine staining: For staining

histamine, we principally foliowed the meth-

ods of Thunberg (1967) and Ehinger and

Thunberg. (1967) with some modifications.

Fresh salivary glands removed in Clarke'ssolution or saline were put on glass slides

and excess liquid were soakcd up with a

filter paper. Then these glands were exposed

to gaseous o-phthalaldehyde (OPA) at 680Cfor 20 seconds. Thev were immediately i -transferred with the glass slides into a rnois-

ture chamber at room temperature for 1.5minutes, then inounted with Clarke's solu-

tien or saline and observed under a fluores-ccnce microscope (Ol>,mpus inodel BH 2-RFL). The presence of histamine was dcter-mined by blue and yellow fluorescencethrough UV and B filters for excitation,

and 430nrn and 530 nm filters for emission,

respectively.

2) Esterase staining: cr-naphthyl acetate

and I3-naphthyl acetate were used as sub-

strates, follosving the inethud clescribed byBurstone (1962),

In vitro tests

Anticoagulation te,rt: Clotting time was

determined bv mixinu O.1 ml fresh mouse . o

blood with O,05ml crude and fractionatedsalivary gland cxtracts, 1'he mixturc was

shaken gently by hand until it ceagulated.

Clotting tinie svas coTllpared xvith thnt of the

control added ",ith salirie.

Hemogysis test: One-twentieth ml crude

and fractionatecl salivary gland extra{'ts were

added to O.2mi of 2C? human red bloodcell (blood type B) suspension in PBS, and

the mixture was observed for hemolysis bya light microscope after O, 30 and 60 minutes

in(;ubation at 370C.

I'inzyrrie test: Esterolytic activities were

tested in vitro wih synthesized substrates,

P-toluensulphonyl-L-arginine methylester

(TAME), N-benzoyl-tyrosine-ethylester-hy-drochloride (BTEE ) , and N-acetyl-L-tyrosi"e-

ethylester (ATEE). Each substrate wfas dis-solved in O.05 M Tris-HCI pH 8.1 containing

O.OlM or O,1 M CaCl.. One-tenth ml sali-

vary gland extract was n]ixed with 2.9m]substrate soluitQn. Changes in absorbanc:e

at 247 nm (TAME>. 256 nm (BTEE}, and

NII-Electronic




318

237nm (ATEE) were monitored for fiveminutes in a spectrophotometer at room

temperature.

Chromatographic analysis

Thin layer chromategraphies (TLC) en

silica gel 60 F254 (Merck Co.) were accem-

plished with twe solvent systems; butanol :acetic acid:H20 (4:1:5>, and ethanol:

25% NH,OH (4:1), Spots were visualized

by spraying ninhydrin solution.

Paper chromatography was performed on

Toyoroshi No, 51 paper, After developmentwith ethanel : O.1 N HCI (1:1) solvent sys-

tem, the chromato-paper was dried and

sprayed with O.1 N NaOH and then 1%･ OPAsolution in xylene. It was observed under an

ultraviolet Iight.

Further ana]ysis of arnines was carried

out by high performance liquid chromato-

graphy (HPLC, Toye Soda HLC-805),Column (4.0× 50mm) was packed with

ion-exchange resin, Toyosoda IEX-215.After applying the sample to the column,

it was eluted with O.25M sodium citrate

buffer (pH 5.28) containing 2 M NaCl. Flowrate was O.5ml per minute. The eluted

amines were reacted with 1% OPA in 1 Mborate buffer (pH 10.5) at the flow rate of

O.1ml per minute. The temperature of the

coluinn was adjusted to 70eC and the reac-

tion was performed at 600C. Ultravioletwave length for excitation was 340 nm, and

emission was monitored at 455 nm,

REsuLTs

Extravasation in guinea Pigs caused by in-

jection of crude extracts

Intracutaneous injections of the crude

extracts were made on the backs of guinea

pigs not sensitized previously with mosquito

antigens. The tests were repeated six times.

As shown in Fig. 1, extravasations were ob-

served at the injected sites with the salivary

gland and midgut extracts of Cx. PiPiensPaltens and the head and thorax extract of

Ae. albopictus after staining with Evansblue. Intensity of these reactions was lessthan that of the positive control where 50 ng

authentic histamine was injected, Erythemaformation was also observed around these

Jpn. J. Sanit, Zool.

Fig. 1 Extravasation of Evans blue in guinea

pig skin induced by mosquito extracts

Sample; S: saline, Him: histamine, Sg:

salivary gland, Cp: crop, Mg: midgut,

HT: head and thorax. Sg, Cp and Mg

were fibtained from Culex Pipiens Pallens and HT from Aedes alboPictus.

Each injected sample involved 4 female

equivalent extract, or 8 salivary grand

equiva]ent.

'llt.

sites. Neither extrava$ation nor erythema

w'as observed at the sitcs injected with crop

extract or saline control, The rnean diameterof the blue spots induced by the injection of

salivary gland extract was 5.3× 4,5mm,From these results, it is eN,ident that the

salivary glands, head and thorax, and mid-

gut contain some chemical substance(s>

which causes extravasation,

Histochemical staining of sativary glands Because histamine and esterases have beenfound in venoms of various arthropods, we

first examined histochemically the possible

presence of these substances in the salivary

NII-Electronic



The JapanSociety ofMedical Entomology and Zoology

Vol. 36 No. 4 1985

glands of mosquitoes.

Hista'rnine staining with OPA: The sali-

vary glands of Cx, pipiens pallens reacted

to OPA and exhibited bluc and yellow fluo-rescence; most parts of the gland were re-

active upon emergence of the mosquito

(Fig, 2a), but fluorescent intensity seerned

to inerease after einergccne. On the fifth

day post-emergence (Fig. 2b}, fluorescenceat proximal portion of the lateral lobes was

a little more intense than that at the distal

portion and the median lobe (Table 1).

The salivary glands of females of Ae.alboPictus ancl Ae. to.aoi were positive forthe OPA reaction. In particular, fluores-ccnce at the proximal portion of the laterallobe of Ae. togoi was very strong.

It is known that saliva can be collected

from the gland by making a shallow cut on

the salivary gland cPoehling and Meyer,19801). So, we fixed the gland of Ae. togoi

319

in methanol and cut it at a site of proximalportion of the lateral lobe, then exposed itimmediately to OPA for observation (Fig.2c, c'). The proximal portion turned to

negative for both blue and yelloMJ fluores-

cence. But, the distal portions of the latera]

and median Iobes that had been severed

off the proximal portion were positive for

fluorescence: while the control gland, fixedwith methanol but unc:ut, stained entirely

positive for the OPA reaction. These results

indicate that OPA reactive histamine hadbeen contnined in the salivary gland and

flowed out of the proxinial portion through

the cuL

Estera,se staining: As shown in Table 2and Fig. 3, esterase activity was definitely

observed in the salivary glands of Cx, piPiensPallens harvested immediately after emer-

gence (Fig. 3a), and on the fifth and twe]fth

days post-en]ergenee ver}r strongly (Fig. 3b).

K$<atkY`:-3

'i'

"T- CFig. 2 OPA reaction of rnosquito sa]ivary

a: Immediately after emcrgunce of Culex

bt Salivary gland on the fifth day post

c and c': Salivary glands of Aedes togoi.

of the lateral lobe and reacted with OPA.

cent microscopic observation of the same

pl: proximal portion of the lateTal lobe, d]:

lobe.

Bars indicate 50 pm.

glands

pipiens patlens.ernergencc of Cutex pipiens pailens. A shallo"' cut was made at proximal

c, Light microscopic observati"n. c',

samp]e with c.

distal portion of the lateral lobe, ni:

portlonFluores-

median




320 Jpn. J. Sanit. Zool.

Table 1 O-phthalaldehyde reaction of mosquito salivary glands for histamine detection

Mosquitoes

Cutex PiPiens Paliens

Immediately after emergence

5 day old

tO day eld

15 day old

Aedes alboPictus

12 day old

Aedes togoi

12 day oldtttt -tttt -t tttttt t

Detection: Excit. UV, Emiss. 430nrn

Excit. B, Emiss. 530nm

Intensity of staining: ± insignificant,

Proximal portion Distal portion of lateral lobe of lateral lobe

Distal portionof median lobe

+--ff

+

-

(blue fluorescence)

(yellow fluorescence)

+ slight, H+ moderate, -

+"-"

+

"

+"""

+

strong, lfF very strong.

"

.e

a-

Pl

!

dl

b

.'1/ .

-- d

Fig. S Nonspecific reaction of esterases in salivary giands a-naphthyl acetate (a, b, and c> and B-naphthyl acetate {d) were used as substrates.

a: Whoie salivary gland oE Culex PiPiens Paltens immediately after ernergence.

b and d: Salivary gland of twelve day old Culex Pipiens Pallens. c: Proximal portion of the lateral lobe of Aedes albopictus.

1: lateral lobe, pl: proxirnal portlon of thc lateral lobe, dl: distal pertion ef the lateral

lobe, m: median lobe.

Bars indicate 50 pm.

NII-Electronic Mbrary



The Japan Society of Medical Entomology and

Vol. 36 No.4 1985

Table 2 Histochemical

Zoology

observation ofesterasesin salivary glands of mesquitoes

321

Mosquitoes Proximal portion of lateral lobe

Distal portionof lateral lobe

Distal portionof median lobe

Culex PiPiens PaUes

Immediateiy after emergence

5 day old

12 day old

Aedes alboPictus

l2 day old

Aedes togoi

12 day old

-l'!nt-tVlntve1ew

+1-

+1+-

-lnt-ll/nt-IW

± 1+

± 1+

+H'lnt-+lntua1-

± 1+

± 1+

a-naphthyl acetate and P-naphthyl acetate were used as substrates.

Numerator; reactivity to a-naphthyl acetate. Denominator; reactivity to flnaphthyl acetate.

Intensity of staining; ± insignifieant, + slight, 'H'

moderate, - strong, l- very strong.

nt; not tested.

Table 3 Results of in vive and in vitro tests with crude and fractionated extacts of salivary

oi Cutex PiPiens Pallensglands

Tests Crudee) Higher mol.

wt. fractiont)Lower mol.

wt. fractiong)Saline

Extravasation testS) {mm)Anticoagulation testb)

Hemolysis teste)

Esterolytic testd) TAME

BTEE

ATEE

6.3× 5.4

2' 3si,

not detectednot detected

not detected

3.4× 2.4

2r 3ot'

nth)

nt

nt

,.iI'g.i

2' 32"

nt

nt

nt

-2'

36',

nt

nt

nt

a): O,05 rn1 of samples was

average of duplicate tests. b> 40 gland equivalent!test. g) :

NNXhen the salivary glands of Ae. alboPictus

and Ae. togoi were stained by a-naphthyL

acetate as a substrate, the proximal portionsof the lateral lobes stained slightly (Fig. 3c),and other portiens insignificantly. When 6-naphthyl acetate was used as a substrate,

whole salivary gland of Cx. Pipiens pallensstained very strongly (Fig. 3d), and in Aedesspecies proximal portions of the lateral lobesreacted moderately.

In vivo studies zvith fractionated salivary

gtand extracts

In the extravasation tests on unsensitized

guinea pigs (Table 3), the crude extract

and LMF caused positive reaction but HMF

led to insignificant result, indicating that

HMF was ]ess important as the cause of

injected intracutaneously

, c), d) : See materials 18 gland equivalentltest.

into guinea pigs not sensitized previously. The

and methods. e) : 20 gland equivalentltest. f) : h): not tested.

extravasation in unsensitized animals.

In vitro studies zvith salivary gland extracts

Anticoagulation and hemolysis tests were

carried out with the crude extract, HMF,

and LMF as shown in Table 3. Mouseblood mixed with each of these samples

coagulated nearly at the same time with the

control that was added with saline. Hemo-lysis of human blood by these samples was

not detected after incubation of O, 30 and

60 min.

Esterolytic activity was not detected when

TAME, BTEE, and ATEE were used as

substrates, indicating that these substrates

might be unsuitable for esterases that might

be contained in the salivary glands of Cx.

pipiens pallens,




322

Chromatographic analysis

The results of TLC of the crude extract

of salivary glands of Cx. Pipiens pallens per-formed with two solvent systems are shown

in Fig. 4. There were spots close to the Rfsof authentic histamine. When tested by

paper chromatography developed with a

mixture of ethanol and hydrochloric acid

and observed under UV light, there was a

spot at around Rf O.75, similar to that for

histamine (data not shown).

To confirm the presence of histamine inthe crude extracts of various parts of the

mosquito body, further analysis was per-formed by HPLC. A HPLC chart (Fig. 5)for the salivary gland extract shows tliatthere was a significant peak of histamine

1

2

HlmSg

ext.

HimSg

ext.

FSg. 4

H,O

Spot

Table

at elution

ologically

e o.2s o.s o.7s 1.o

Rf.

Thin layer chromatograms of sallvary

gland extracts of Culex Pipiens PallensSelvent system: 1, Butanol:Acetic acid:

(4:1:5). 2, Ethanol: 25% NHsOH(4:1). staining: Ninhydrin.Him: histamine, Sg ext.: salivary glandextract,

4 Amount of amines in the body parts of CJe.Iiquid chromatography

ggw:uvB8m

. tlrneactive

Spd

Put

Hi

around .amlnes'

Jpn. J.

5.2 min.such as

Sanit. Zool.

Other bi-

putrescine,

Celumn resin! IEX-215

size: 4 X SO rnTa

tempe=ature: 70 oC

EIuent: Sodium citrate buffer

Detection: OPA reactien

Reaction ternperature: 60oC

Excitation: 340 nm

Emisslon! 4SS nn

spm

,5HT S iO l5 20 2S

Retention time ( min. )

Fig. 5 High performance liquid chromato-

graphic separation of amines in salivary

glands of Culex pipiens paUens Put: putrescine, Him: histamine, Spd:

spermidine, Spm: spermine, 5-HT: sero-

tonin.

PiPiens PaUens anal)rzed by high performance

Sample Him Put Cad Spm AgrnSpd 5.HT

Whole bodyHead and Thorax

Abdomen

Salivary glandHead

10. 28 ( 3. 65)8.58 ( 7.27}-(-)

2. 42 (80. 6 )6.81 (45.4)

134. 945.

216.3

5.4

10. 5

5. 36

8. 49

225. 48S.1214.332.2

15.4

3.10 -9.19

8. 354. 97S.

991.

451.37

-

-

Arnine amounts are expressed as nglind

of histamine (ptglwet weight g) .

-: not detected.

Him: histamine, Put: putrescine, Cad:midine, 5-HT: serotonin.

ividual. Figures in

cadaverine, Spm:

parentheses indicate the concentration

spermine, Agrn: agrnatine, Spd; sper-




Vol. 36 No.4 1985

spermidine, and spermine, were also detected

at about 4, 7, and 14min, respectively. 5-

Hydroxy tryptamine (24min) was not de-

tected by the present system applied. An

unidentified peak was observed at about 13.8

min that is slightly earlier than that of sper-

mine. The amount of histamine and other

amines detected by HPLC in the crude ex-

tracts of the whole body, head and thorax,

abdomen and salivary gland of female mos-

quitoes' are shown in Table 4. Histamine

was detected in all the extracts except for

abdomen, large portion of histamine being

found in the extracts of salivary gland and

head,

DiscussroN

The paired salivary glands of the mos-

quito are located anteriorly within the

thorax. Each gland is composed of three

lobes, two lateral and one median. Each

Iateral Iobe consists of two glandular regions,

or proximal and distal portions, which are

subdivided by non-glandular, intermediary

portions. The rnedian lobe has non-glandular

and glandular portions. The salivary duct, or central canal, runs the length of each

lobe. These Iateral ducts fuse with one an-

other as they emerge from the lobes, and

run forward laterally to fuse into a common

duct in the neck and head. This common

duct enters the salivary pump at the base

ef the hypopharynx (Clements, 1963). Sec-

retory material which appears in glandular regions as large secretion masses is entirely

extracellular and seen in the central canal,

periductal space or apical cavity (Wright, 1969). Such salivary secretion is voided

during blood feeding (Barrow et al., 1975),

passing through the common duct in the

head region. The salivary secretion is re-

sponsible fer the skin reaction in host ani-

mals, because when the main salivary ducts

at the neck region of a female mosquito

were cut, no reaction occurrecl on the skin

after her bite on a man whe was sensitive

to the bites of normal mosquitoes (Hudson et al. 1960). Many authors observed

patholog:,cal reactions when extracts of mos-

quitees' salivary glands were injected into

man's skin. There is, however, still ver}r lit-

323

tle information on the components of the

salivary glands. The presence of histamine has Iong beensuspected but remained uncertain, In this

study, crude extract of the sa!ivary glandsef Cx. PiPiens pallens showed extravasation

activity in guinea pigs that had not beensensitized previously (Fig. 1). The activity

was found in the Iower molecular-weight

fraction of the salivary gland extracts ob-

tained by SHFC (Table 3). The positiN'eresults of histochemical studies on the sali-

vary glands and HPLC analysis definitely

proved that histamine is involved in the

salivary glands (Fig. 2 and 5, Table 1 and

4). Eckert et al. (1950, 1951) preparedextracts from the whole body, head, middle

part with legs, hind part, and wings of Cx.

PiPiens. But they did not deal with the

salis,ary glands separately. Their extracts,

except that from wings, caused contraction

of guinea pig intestine, which was inhibitedby histaminase treatment. They reported

that the head and the middle part contained

O.157 of histarnine, and that ca. 40% of

which were lost after blood feeding. But

McKiel (1959) suggested that there was

neither histamine nor histamine-like com-

pounds in Ae. aegyPti. He found no positivereaction to the bite of the insect on animals

that had not previously been exposed to

mosquitoes. Even when he injected whole

body extract of Ae. aegyPti, he observed no

[`true'] skin reactions. These conflicting re-

sults rnight be due to species difference of

mosquitees, since our histochemical observa-

tions revealed strong OPA staining in Csc.

PiPiens Pallens and Ae. togoi, but relatively

weak reaction in Ae. alboPictus. Our results,

however, conclusively demonstrated hista-･mine in the salivary glands of mosquitoes･ --for the first time. Most of histamine in-

volved in the female mosquito of Cx. pipienspallens was detected in the salivary glands,and head (Table 4). But we still do not

know abeut the exact distribution or locali-

zation of histamine in the head region.

Saliva of the mosquito is carried to the

mouth parts passing through the common

duct in the head. We suppose that at leasta part of histamine in the head may exist in

such duct. The amount of histamine de-




324

tected in the present work was less than

that reported by Eckert et at. (1950, 1951).The difference might be to seme extent dueto extraction eMciency or methodologica] dif-ference applied.

By HPLC analysis, we detected polyaminessuch as putrescine, spermine and spermidine

in the salivary glands of mosquitoes (Table4). It is noteworthy that serotonin was un-

detectable in any part of this insect by the

method applied in the present study. Poly-amines are commonly found in many kindsof cells. We suppose that such polyaminesweuld play physiological roles in the sali-

vary glands of mosquitoes.

Positive reactions to esterases were ob-

served throughout the salivary glands of Cx.

PiPiens paltens when a- and B-naphthylacetates were used as substrates. But the

salivary glands of Ae. albopictus and Ae.togoi reacted slight]y to a-naphthyl acetate

and moderately to P-naphthyl acetate at

proximal portion of the lateral Iobe (Table2). In AnoPhetes stePhensi, Poehling and

Meyer (1980) demonstrated positive reac-

tions by the azo dye staining method in thewhole salivary glands, especially in the apical

cavities of epithelial cells and in the lumenof glandu]ar parts. They noted that esterases

of An. stePhensi were reactive to or-naphthylacetate but, contrary to our results, not to

P-naphthyl acetate. Therefore, it seems like-]y that there are species difference in thecomposition of esterases among the salivary

glands of different species of mosquitoes.

Poehling and Meyer (1980) also suggested

the possibility that these esterases might con-

tribute to dilate subepidermal capillaries in

animals. But, our results obtained with Cx.

Pipiens pallens do net support their view,

since the higher molecular weight fractionof the salivary gland extract, which are

considered to contain esterases, did not cause

extravasation in guinea pigs. Tests for esterolytic activity with synthe-

/sized substrates, TAME, BTEE and ATEE,

were all negative (Table 3), although the$e･substrates

are known to be very sensitive in/detecting trypsin- and chymotrypsin-like en-

zymes. Metcalf (1945) also reported that

extract of the salivary glands of AnoPhetes

guadrimaculatus was negative for proteases

Jpn. J. Sanit. Zool.

when casein was used as a substrate. Raoand Subrahmanyam (1969, 1970) found

phospholipase activity in the body of Cx.

Pipiens fatigans, Ae. aeg){Pti and An. stePhen-

si. Geering (1975) considered that hemo]y-tic activity observed in the midgut of blood-fed Ae. aegypti was due to phospholipasesecreted from the gut epithelium. We didnot observe hemolysis with salivary glandextracts, so that such a toxic constituent may

not be contained in the salivary gland of

Cx. Pipiens Pallens. Metcalf (1945) also

considered that phospholipase A is not prob-ably present in the salivary secretion of An.

quadrimaculatus.

In our studies with the salivary glandextracts of Cx. Pipiens pallens, only histamine

was the sole substance which e]icits the

immediate skin-reactions. rlhe

higher mo-

lecular weight fraction of the salivary glandsof Cx. PiPiens Pailens did not cause extra-

vasation in guinea pigs, but when we carried

out prick tests in two persons, who hadexperienced strong reactions to mosquito

bites, erythemas with sizes more than 20 ×

15mm developed within 15min. This is

probably due to an antigenic property in-volved in the salivary glands as considered

by many authors (Mellanby, 1946; Gordonand Crewe, 1948; McKiel, 1959; Allen and

West, 1966). An antigenic protein isolatedfrom the oral secretion of Ae. aegypti had

a mol. wt. of about 33,OOO (Newsome et at.,

1969). Willadsen and Williams (1976) and

Willadsen (1976) reported that, in BoophilusmicroPlus, an esterase causes an immediatehypersensitivity reaction in cattle by its anti-

genic property but not by enzymatic proper-ty. The esterases or other higher molecular

substances in the salivary glands of mosqui-

toes may act as antigens which may lead tohypersensitivity in man. Further studies will

be necessary to elucidate the antigenic

properties or enzyrnatic activities of these

esterases.

AcKNOwLEDaEMENTs

The authors express their deep gratitudes to

Professor A. Spielman, Department of TropicalPublic Health of Harvard University, for hiscritieal reading of the first manuscript; to Dr.H. Itokawa, Tokyo Medical and Dental Univer-

NII-Electronic




Vol. 36 No.4 1985

sity, and Prefessors T. Shigei, Y. Nagai and T.Yoshida, Nagoya University School of Medicine,for critical discussions and encouragement.

REFERENCES

AIIen, J. R, and A. S. West (1966); Some

properties of oral secretion from Aedes aegrPti

(L.). ExP. Parasitol. 19: 124-131.

Barrow, P. M., S. B. Mclver and K, A. Wright

(1975): Salivary glands of female Culex

PiPiens; ptforphological changes associated with

maturation and blood feeding. Can. Entomot.,

107: 1153-1160,Burstone, M. S. (1962): Enxyme histochemistry

and its apptication in the study of neoPlasms,

621 pp., Academic Press, New York.

Clements, A. N, (1963): The physiology of

mosguitoes, 393pp., Pergamon Press, Oxford.Eckert, D., M. Paasonen and A. Vartialnen

<1950): On histamine in gnat (Culex Pipi- ens). Ann. Med. ExP. Biol. Fenn. 28: 84-85.Eckert, D., M. Paasonen and A. Vartiainen

(1951): On histamine in the gnat (Culex piPiens). Acta Pharamocol. Toxicol. 7: 16- 22.Ehinger,

B. and R. Thunberg (1967): Induc-

tion of fluorescence in histamine-containing

cells. ExP. Cell Res. 47: 116-122.Feingold, B. F., E. Benjamini and D. Michaeli

(1968>: The allergic responses to insect bites.

Ann. Rev. Entomol. 13: 137-158.Geering, K. (1975): Haemolytic activity in the

blood clot of Aedes aegyPti. Acta TroP. 32: 145.151.

Gille, J, D. (1971): Mosguitos, 274pp. Wei-

denfels and Nicolsen, London.Cordon, R. M. and W. Crewe (1948): The

mechanisms by which mosquitoes and tsetse-

flies obtain their blood meal, the histology of

the Jesions produced, and the subsequent re-

actions of the mammalian host; together with

some observations on the feeding of Chrysops

and Cimex. Ann. TroP. Med, Parasitol,, 42: S34-356,

Hudson, A., L. Bo"'man and C. W. M. Orr

(1960): Effects of absence of saliva on blood

feeding by mosquitoes, Science, 131: 1730- l731.McKiel, J, A. (1959): Sensitization to mosquito

bites. Can. J. Zool. 37: 341-351.McKiel, J, A. and J. C, Clunie (1960): Chro-

matographic fractionation of the non-dialyzable

portion of mosqulto extract and intracutaneous

reactions of mosquito-bite-sensitive subjects to

the separated components. Can. J. Zool.. 38: 479-487.

325

pt{ellanby, K. CI946): Man's reaction to mos-

quito bites. Nature, 158: 554,

Mctcalf, R. L. <1945): The physiology of the

salivary glands of AnoPhetes quadrimaculatus, L Nat. Malan Soc. 4: 271-278.Newsome, W. H., J. K. N. Jenes, F. E, French

and A, S. West (1969): The isolation and

properties of the skin-reactive substance in

Aedes aegyPti oral secretion, Can, J, Biochem., 47: ll29-1136.Orr, C, W. M., A. Hudson and A. S. West

(1961): The salivary g]ands of Aedes aeg),Pti.

Histolegical-histochemical studies. Can. J. Zool. 39: 265-272,

Poehling, H. M, and W. Meyer <1980) : Esterases and glycoproteins in the salivary glands of

Anopheles stephensi. Insect Biochem. 10: 189-

198,Rao,

R. H. and D. Subrahmanyam (1969): Characterization ef phospholipase B of Culex

piPiens fatigans. J. LiPid Res., 10: 636-641.Rao, R. H. and D. Subrahmanyam (1970): Distribution and properties of phospholipase A

of Culex PiPiens fatigans. Arch. Biochem. Bio-

PhJ,s., 140: 443-449.

Rock"'el], E. M. and P. Johnson (1952): The insect bite reaction. II. Evaluation of the al-

lergic reaction. J. Invest. Derrnatol.. 19: IB7-

155.Suzuki, S., K. Negishi, S. Tomizawa, M. Shiba-

saki, T. Kuroume and T. Matsumura (1976): A case of mosquito allergy. Acta Allergol,, 31: 428"441,

Takahashi, C., M. Uehara and H. Nomura (1973) :

The allergic response to the mosquito. Jpn. J. Ctin. Derm. 27: 423-429.

Thunberg, R. (1967): Localization of cells con-

taining and forming histamine in the gastric

mucosa of the rat, ExP. Cell Res., 47: 108-115,Urisu, A., C. Sato, J. Okada, H. Matsuoka, M. Matsuoka, S. Torii and A. Koda (1981): Im-

munological stuides of the two patients with

mosquito allergy. Clin. Immunol. 13: 499-

508.Willadsen, P. (1976): Allergenic activity of an

esterase frem BooPhilus microplus. FEBS Lett.,

72: 346-349,

Willadsen, P. and P. G, Williams (1976): Ise-

Iation and partial characterization of an antigen

from the cattle tick BoePhitus microPlus. Im- munochemistry, IS: 591-597.Wright, K. A. (1969): The anatomy of salivary

glands of Anopheles stePhensi Liston. Can. J. Zool. 47: 579-587.Yasuda, K, and K, Hirano (1980): A case of

mosquito allergy, Jpn. J. Clin, Derm., 34: 293-297.

NII-Electronic



The 　Japan 　Sooiety 　of 　Medioal 　Entomology 　and 　Zoology

326

摘　　要

蚊の唾液腺のヒスタミンおよび

　　エステラーゼについて

蚊刺咬による皮膚反応の機構を解明する試みのひと

つとして，蚊の唾液腺に含まれる成分について分析し

た・アカイエカ Culex　pipien∫ Pallensの唾液腺の粗

抽出物およびその低分子画分（mo1 ．　wt ．＜6，000＞は，

蚊に無感作のモルモット皮内で血管透過性を亢進し

た．0 −phthalaldehyde 法により histamine染色を行うと，Cx ．　pipiens　Pallensおよび Aedes 　togei の唾

Jpn．　J．　Sanit．　Zool．

液腺全体に強い陽性反応が観察された．α および βナ

フチルアセテー

トを基質としてエステラーゼ染色を行

ったところ，Cx．　 pipiens　PalJensの唾液全体に強い

陽性反応が認められ，一方， Ae．　togoi および Ae・albopictus では比較的弱い反応が観察されたのみであ

った・CX ．　pipiens　PaUens の唾液腺抽出物を用いた

in　 vitro 実験では，　 TAME ，　BTEE および ATEE の

加永分解，血液凝固阻止活性および溶血活性は検出さ

れなかった．イオン交換高速液体クロマトグラフィー

分析では，（7x．　 pipiens　Pallensの唾液腺抽出物中に

histam三ne ，　putrescine，　 spermine ，および spermidine

が検出されたが，serotonin は検出されなかった，

N 工工一Eleotronio 　Library 　

histamine in culexthe

Documents