Jpn. J. Infect. Dis., 52, 214‑216, 1999

Short Communication

Plasmodium Infection‑Induced Changes in Salivary Gland Proteins ofMalaria Vector Anopheles stephensi (Diptera: Culicidae)
Harish Shandilya, S.K. Gakhar*and T. Adakl
Department ofBiosciences, Maharshi Dayanand University,Rohtak‑124 001, Haryana and LMalaria Resewch Centre, New Delhi‑110 009, Ihdia(Received January 18, 1999. Accepted September 24, 1999)
ヽ∫SUMMAkY: parasitism by Plasmodium yoelii yoelii induced 18 polypeptides in the salivary glands of aglng●malaria vector Anopheles stephensi. A polypeptide of low molecular size (30 kDa) Could generally be induced at
all infected stages. On day 5

post blood feeding (PBF), no new polypeptide could be found in the salivary

glands･ Seven polypeptides of low molecular size and 3 0f high molecular size could be induced on day I I PBF,

which inducibility coincided with the invasion of the salivary glands by the sporozoites. Quantitatively, Soluble
proteins decreased in the salivary glands by about one‑third in females that had consumed infected or uninfectedblood meal on day 9 (oocysts stage) as compared to nonfeeding females. However, on day 15, in the salivary

glands invaded by sporozoites, the amount of proteins obtained from infected females was approximately 26%
lower than that obtained from uninfected females. A similar reduction was also observed in aged (20 days PBF)
Saliva亘glands of infected mosquitoes. These proteinsCould confer parasite tolerance to the females and enhanceparasite transmission potential.

Mosquito salivary glands often harbor viruses and proto‑ites were observed in salivary glands 10‑ I 1 days PBF. Control

zoan parasites that infect vertebrates. During blood feedingmosquitoes were fed on uninfected mice･ Fully engorgedand probing, salivation provides the means for transmitting

females were divided into three groups/replicates, and thethese pathogens to the new host ( 1 ). Mosquitoes infected withsalivary glands were removed from about 60 individuals in●Plasmodium exhibit behavioral and physiologlCal changes,

total. Salivary gland samples were taken for analysts On days

which include adversely affected blood feeding, fecundity,l (12h PBF), 2, 5, ll, and20PBF. One sampleofnonfeeding

and longevity, etc. (2). Limited information is available

female salivary glands (0‑days old) and one sample of sugar‑

regarding the biochemical changes in the vector Anopheles

fed female salivary glands (4‑days old) were taken as control･

after Phzsmodium infection. Hogs et al. (3) reported a reduction

Salivary glands collected from mosquitoes at the different

in the accumulation of protein content in the ovaries, while

stages mentioned above were individually homogenized in

Vanderberg et all (4) found a lesser synthesis of amino acids

60 LLl of phosphate‑buWered saline (PBS) (pH 7･4) with prDteaSein the hemolymph of infected mosquitoes. Manytypes of host‑inhibitor, 1 mM phenylmethylsulfonyl nouride (PMSF), on

parasite interaction have not been studies in any great detail･It remains to be seen whether the genes that express in salivary

centrifugation as described by Vanderberg et all (6)･ The

glands or their products could be the targets for future vector
control strategleS･ In the present investigation, an attempt hasat 40C to remove salivary gland debris. The supematant wasbeen made to study the malaria parasite infection‑induced

centrifuged agaln at 1000 g for 15 min at 4oC･ The parasites

ice. The parasites from infected tissues were removed byhomogenates were centrifuged slowly at 50‑100 g for 5 min●●changes in the qualitativeand quantitative pattems of polypep‑

were concentrated in the pellet, then the supernatant wastides in the salivary glands of Anopheles stephensi, a vector

confined to be free from parasites by Leishman's strain (7)･of the malaria parasite on the Indian subcontinent.

Centrifugation was carried out agaln if parasites were present●The Delhi strain of A. stephensi was maintained at 28 ± 2oC

in the supernatant･ After a final centrifugation at 10,000 g

and 80% RH (5) in our laboratory. The mosquitoes infected

for 15 min at 4oC, an equal volume of sample buffer for sdiumwith the malaria parasite, Plasmodium yoelii yoelii (265 strain

dodecyl sulfate polyacrylamide gel electrophoresis (SDS‑by INSERM, Paris, France), were reared at 24℃ until use.

PAGE) (8) was added to the supernatant. The quantity ofBALB/c mice (5‑6 weeks old) were infected by intraperitoneal

soluble proteins in normal and infected salivary glands wasin∝ulation of heparinized blood obtained from a donor mouseestimated by the method of Lowry et al (9)･

infected with P.y.yoelii. About 0.5 ml of infected blood from●

The qualitative changes in the salivary gland proteins in A･●

these donor mice at 4 days after prlmary Inoculation resulted

Stephensi due to Plasmodium infection are shown in Figurein the production of ex flagellating gametes after 36 h in the1. More than 30 polypeptides were observed in the salivary

recIPlent mice. Mosquitoes were allowed to feed on these●glands during the adult life of the control females, which had

fed on uninfected mice. Eighteen additional polypeptides weremice at 48 h posトinoculation.

About 100 4‑day old females were not allowed sugar

obtained from females that had fed on infected blood. The

feeding for 12‑ 18 h before blood feeding on mice･ Nonfeeding

polypeptide with the molecular size of 30 kDa was induced

mosquitoes were discarded･ More than 1 00 oocysts developed

generally in all the adult stages when the mosquitoes had fed

on the midgut 4‑5 days post bl∝d feeding (PBF),and sporozo‑

on infected blood, excepting some stages. On the first day,*Corresponding author: Fax: +91‑1262‑41132, E‑mail:molecular size polypeptides of 26, 32･5, 50, and 82 kDa were

12 h PBF, no new polypeptide was found･ However, 4 low

induced in the salivary glands at 2 days PBF. At the oocyst‑

gakhar@ mdul. emet. in214

M UF GF P C P C P C P C P C
O‑D4‑D

1‑D

2‑D

5‑D

11‑D

20‑D

POST BLOOD FEEDING
Fig･ 1 ･ The changes of polypeptides in the salivarygland ofA stephensi
infected with P. y. yoelii.

UF =Unfed GF =Glucose fed
C=Controlled P

day=Parasitized

0day4

M =Molecular weight markers
D

day5dayll

day20

Post blood feeding=Days

Fig･ 2･ Protein contents in salivary glands of A･ stephensi

positive stage, 5 days PBF, no new polypeptide was found in
the salivary glands. After the salivaryglands had been invaded

at various stages, as described above. This finding could alsoby sporozoites (day ll PBF), 7 low molecular sife (14･5, 21,indicate that now of salivary proteins is continuous throughout

23, 63, 65, 72, and 82 kDa) and 3 high molecular sIZe pOlypep‑

blood‑feeding. Ponnuduraiet al. (14) demonstrated that saliva‑

tides (147, 151, and 153 kDa) were induced. The density of
2 polypeptides (37･5 and 40 kDa) was enhanced. In agedtion occurs, although irregularly, throughout blood‑feeding･The highly conserved stress response of parasitism involves

salivary glands (20 days PBF), only 2 polypeptides (44.5 andshutting off the general transcription and activating the tran‑

55 kDa) were induced.

scription of a selected set of genes co°ed fわr the heat shock

These results suggest that infection of mosquitoes by para‑proteins (HSPs) or stress proteins (15)･ This 乍ould explain thesites alters the quality and quantity of specific polypeptides

synthesis and disappearance of several protelnS after parasite

in the salivary glands and results in enhanced transmission

i nfection.

of parasites･ The dynamics of transmission of many arthropod‑

Quantitatively, soluble proteins were found to be 3･2 LLgper

borne pathogens are related directly to the phase (probing,palr Of salivary glands in freshly emerged nonfeeding female

engorgement,and withdrawalof mouthparts) of blood feeding.

mosquitoes. This level decreased by 34% (P < 0.01) in the●Kelly and fklman (10) reported that mosquitoes salivate andsalivary glands of 4‑day old glucose‑fed females, and bytransmit sporozoites during probing, before engorgement withblood. A study by Li and Rossignol (I 1) found that salivation

66% in blood fed females (control) of corresponding age (Fig.
2). On day‑9 (5 days PBF), when oocysts were observed in(transmission of parasites) ∝curs pri.mari1y during probing, andthe midgut, Soluble protein content was not found to havealso demonstrated that later transmlSSion is inversely propor‑
tional to apyrase level. Increased probing time possibly resultschanged sigmificantly in control females (l･ 16LL gper Salivarygland pair) or in infected females (1 ･ I 3 LLgper Salivary glandin release of more sporozoites (12). The invasion of parasitespair). After the second gonotrophic cycle, on dayl 15 (1 l days

causes cellular damage in host salivary glands. Interestingly,PBF), the quantity of soluble proteins was almost doubled

this does not effect salivation during probing. Studies on theinfection of Plasmodiumfalciparum ( 1 3) found no difference

females. However, in infected females, sporozoites presented

in the amount of saliva secreted between infected andin the salivary glands,and qu弧tityof soluble proteinswas about

uninfected mosquitoes.

26% less than that in control females (P < 0.01). Similarly,
on day‑24, the quantity of soluble proteins was about 23%
less (P < 0.01) in infated females salivaryglands thanthat in(2.60LLg) compared to that on day‑9 (P < 0･01) in control

Our study demonstrated a depletion in salivary glandproteins after the females had fed on infected blood meal.
This finding could be ascribed to the disappearance of 5 low

salivary glands of the control.
●molecular size polypeptides (19.5, 29, 31, 34, and 66 kDa),

It was also observed that, comparatively, females havlng

after exposure to infected blood meal at various respective

stages of adult life. On the first PBF day, 3 polypeptides (29,sporozoites in the salivary glands and00cysts in the midgut
did not take a smaller amount of blood meal (unpublished31, and 66 kDa) disappeared. On day 5 and day 20 PBF, adata) than other groups did. Thus, the reduction ln protein

19.5 kDa polypeptide was found in control females, though notcontent in the salivary glands of infected mosquitoes must bein infected females. The 34 kDa polypeptide also disappeared

due to the presence of parasites rather than to poor quality ofat an aged stage (20 days PBF). However, the density of 4
low molecular weight polypeptides (23, 26, 37.5, and 48 kDa)that the percentage of depletion was 13 times greater whenblood meal･ This proposition is further supported by the fact

was reduced in relation to age after the third gonotrophic cycle.

sporozoites were infecting the mosquitoes salivary glands thanHowever, this depletion also coincided with the induction of

in oocysts‑infected mosquitoes･ This finding could also be

new polypeptides of malaria‑infected anopheline mosquitoes

attributed to the diversion of nutritional resources from the215salivary glands to the immune system. It is also unlikely that

antisporozoite antibodies in r∝1ent malaria and its rela‑this is due to nutrient competition between the parasites andtionship to protection immunity. Proc. Helminthol･ Soc°,

Washington, 39, 5 14‑525.

mosquitoes, which could cause the reduction in the total

7. Choudhary, D. S. and Ghosh, S. K. (1982): Staining ofsprozoites from infected mosquitoes. Indian･ J･ Malariol ･ ,salivary gland protein content. Because we observed that,

although the percentage of difference in total protein content●19, 143‑144.

decreased in aglng Salivary glands, the infected group never

8. Lammeli, U. K. (1970): Cleavage of stmctural proteinsattained the same level of protein content as that found in theduring the assembly of the head of bacteriophage T 4･control salivary glands.

The synthesis of parasite‑induced polypeptides may be

Nature, 227, 680‑685.

considered a desimble feature inthat it confers paluite tolerwce

9. Lowry, 0. H., Rosenbrough, N. ∫., Farr, A･ L andto the adult･ In conclusion, infected mosquitoes may have aRandall, 良. ∫. (1951): Protein measurement with thegreater potential for parasite transmission because sporozoites

Folin phenol reagent. ∫. Biol. Chem･, 193, 265‑275･

are trZLnsmitted via secretory proteins releasedfrom the salivary

10. Kelly, R. and Iklman, R.D. (1992): Multiple transmission+Iof Plasmodium gallinaceum (Eucoccida: Plamodiidae)

glands.

during serial probing by Aedes aegypti (Diptera: Culicidae)

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