Anim. Behav., 1967,15,336-341

A CHEMICAL FACTOR THAT STIMULATES OVIPOSITION BY CULEXTARSALIS Coquillet (DIPTERA, CULICIDAE)

BY ANNE HUDSON & J. McLINTOCK*Entomology Research Institute, Canada Department of Agriculture, Ottawa, Ontario, Canada

The ways in which a female mosquito detects asuitable site for egg-laying are not yet fullyknown although there is a great deal of evidenceto suggest that eggs are not laid indiscriminately(Buxton'° & Hopkins, 1927 ; Bates, 1949; Muir-head-Thomson, 1951 ; Wallis, 1954) . Such feat-ures of a natural environment as water move-ment, shade and colour, emergent vegetation,salinity and pollution have been associated withthe oviposition habits of different species(Muirhead-Thomson, 1951 ; Detinova, 1936 ;Russell & Rao, 1942 ; Wallis, 1954 ; Gjullinet al ., 1965) ; however, little attention has beengiven to the possibility that developing stagesof a species, particularly pupae and pupalexuviae, are themselves an important character-istic of a successful oviposition site . Such anindication could be detected by the female as avisual, tactile or chemical stimulus acting singlyor in succession and having a cumulative effect .In this paper evidence is presented of a

chemical factor associated with the developingstages of Culex tarsalis which influences thechoice of an oviposition site by females of thisspecies .

MethodThe Culex tarsalis colony maintained in

Ottawa was obtained from the Veterinary andMedical Entomology Unit of the ResearchStation at Lethbridge, Alberta, in 1961 . Themosquitoes were reared and the experimentscarried out in a controlled rearing room main-tained at 75°F and 75 to 80 per cent relativehumidity, with a photoperiod of 16 hr to whichsunrise and sunset periods of 50 min each wereadded.Routine Rearing Procedure

Adults were maintained in cages 2 x 2 x 2 ftand were supplied constantly with 5 per centsucrose in distilled water. Females were fedon young chickens which were put into the cagesovernight on 2 nights of every week . Egg-raftswere laid in Pyrex crystallizing dishes, 190 x 100*Present address : Research Station, Canada Departmentof Agriculture, Saskatoon, Saskatchewan .

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mm. Two laying dishes were kept in a cage, onecontaining pupae in distilled water and theother distilled water only . The eggs were laidovernight and collected on the following morning .During the 2 following days they were examinedfor eye spots to determine whether they werefertile, before being put into rearing trays .Two or three rafts were put into each tray,containing 2 . 5 1 of Bates' `Medium S' (Bates,1941), and the larvae were fed on alternatedays on 0 .25 g of ground rabbit chow mixedwith 0 .04 g of bakers' yeast. Pupae were collecteddaily and put into a bowl as described above .Adults were allowed to emerge directly into thecage. Under these conditions the eggs hatchedin 21 to 3 days and the larval period occupied10 days. Adults would take a blood meal 48 hrafter emergence, although some took longerthan this ; presumably this depended upon therelationship between the time of emergence andthe availability of the chicken . The mediantime for the oocytes to complete developmentwas 5 days after feeding and the minimum timewas 3 days. Oviposition took place between 7and 10 days after feeding .Experimental Procedures

Two different procedures were followed whenoffering test solutions for oviposition .A. Two laying dishes, one containing test

solution and the other a control solution, wereput into the cages. Egg rafts were removed eachmorning and the positions of the dishes werereversed daily over 4-day periods . After eachsuccessive 4 days the solutions were renewed .This method afforded a long series of observa-tions with few rafts laid per night, but it had thedisadvantage of allowing time for changes tooccur in the test solutions and of obtaininginformation from adults of varying ages .B. All laying dishes were removed from a cage

containing females newly fed on blood . Sevento 8 days later a choice of solutions (as above)was offered to the gravid females, most of whichlaid their eggs during the following night. Eggrafts were removed the next morning and thedishes were usually offered again on the second

HUDSON & McLINTOCK: CHEMICAL FACTOR ° IN, OVIPOSITION

night, in reversed positions, to collect the feweggs developing later . Using this method a largenumber of rafts could be obtained during onenight and a group of females within known agelimits was choosing between fresh solutions .

Preparation of Test Solutions1. Pupae in distilled water. Pupae were re-

moved from the rearing trays each day andwashed with distilled water . They were then putinto a laying dish containing 500 ml of distilledwater. The control used for these tests was asimilar dish containing 500 ml of distilled water .In a few trials pupae were put into a 0 .03Msolution of NaCl and in these cases NaClwas used in the control dish .

2 . Emergence water. In early experiments 250ml of distilled water, to which pupae had beenadded daily over a period of _4 days and fromwhich adults had emerged, was filtered throughWhatman No . 1 paper and then offered togravid females with a control dish of distilledwater. In later experiments the emergence waterwas obtained- by allowing a known number ofwashed pupae to inhabit a small volume of glass-distilled water for 24 hr. The pupae and anyexuviae present were then filtered off and thefiltrate was evaporated under vacuum ; in somecases the evaporated sample was restored with asmall volume of distilled water and then freeze-dried. The final dry sample was restored to therequired volume with distilled water and offeredwith a control dish of distilled water . In a fewtrials 0 .03M NaCl was used instead of distilledwater, both to reconstitute dried material and asa control .

3 . Pupal water. This differed from emergencewater in that it had contained only pupae fromwhich no adults had emerged up to the time offiltration .

4. Larval water. Three hundred late 4th-instarlarvae were removed from the rearing trays andwashed with distilled water . They were put into250 ml of distilled water for 24 hr and thenfiltered off. The filtrate was offered in a layingdish together with emergence water which hadbeen obtained in the same manner.

In Table I both the total number of rafts laidand the number of days on which dish 1 re-ceived more rafts than dish 2 were used ascriteria to determine whether a preference wasshown for one dish over the other. The possibilitythat an indicated preference for one dish ° was infact a reflection of aversion to the other was in-

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vestigated by using both distilled water and a0 .03M solution of NaCl (shown to be acceptableto CC . molestus females by Hudson, 1956) asmedia for pupae and as controls .

In cases where experiments were continuedfor 15 or more days, probabilities were calcu-lated using the Wilcoxon signed rank matchedpairs test (Siegel, 1956) . Probabilities were notcalculated for smaller numbers of tests sincecalculations based on n<15 would have beenoutside the estimated limits of precision .

ResultsThe results of experiments I to XI, in which

C. tarsalis females could discriminate betweentwo laying dishes, are given in Table I . Inexperiment I a dish containing pupae in distilledwater received more rafts than a control dishcontaining only distilled water. In experiment IIa dish containing pupae in NaCl also receivedmore rafts than the control dish containing onlyNaCl solution. Since both experiments showedthe same trend it seemed likely that the prefer-ence shown was associated with the presence ofthe insects rather than with avoidance of thecontrol dish .

In experiment III emergence water receivedmore rafts than distilled water . Therefore, itappeared that an ovipositional stimulant re-mained in the water even after removal ofpossible visual and mechanical stimuli resultingfrom the movement of insects at the surface .It was considered that any remaining stimulusmust be of chemical origin and might be eithervolatile or dissolved in the water . In the follow-ing experiments attempts were made to removevolatile components.

A 250 ml sample of C. tarsalis emergence waterwas boiled until 125 ml remained . This samplewas restored to 250 ml with distilled waterand gravid females were offered a choice betweenthis and an untreated sample of emergencewater. The results of experiment IV indicatethat boiled emergence water was preferred .In ° experiment V a similar sample of emergencewater was evaporated to dryness and the residuewas redissolved in 250 ml of 0 .03M NaCl.This material was tested with a control dishcontaining 0 .03M NaCl . Tests with distillatesobtained at 4'C and -75•C confirmed that theattractive material remained in the residue afterflash-evaporation ; however, very small numbersof rafts were laid in these waters . It was con-cluded that' the ovipositional stimulus remainedin the absence of volatile components .

338

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HUDSON & McLINTOCK : CHEMICAL FACTOR IN OVIPOSITION

To determine whether there was any specificcomponent in the preference demonstrated, C .tarsalis emergence water was compared withwater obtained in the same manner from pupaeof three different species ; Culiseta inornata(Williston), Aedes aegypti (L.) and Culex pipiens(L.) . The results of experiments VI to VIIIindicate that C. tarsalis females preferred waterwhich had contained their own species .

In experiment IX the possibility that one sexwas responsible for the stimulus was examinedby allowing gravid females to oviposit in dishescontaining either female or male emergencewater. During the first night twenty-seven raftswere laid in each dish and on the second nightnineteen and twenty-one rafts were laid in themale and female dishes respectively. It was con-cluded that the females did not discriminatebetween male and female samples .

Gravid females were offered a choice betweenC. tarsalis 4th-instar larval water and emergencewater in experiment X. The results indicatedthat both dishes provided the required ovi-positional stimulus. Although the larvae werenot fed during the 24 hr prior to filtration, thewater was undoubtedly contaminated by ex-cretory products .

In experiment Xla, water which had containedonly C . tarsalis pupae (pupal water) was com-pared with water which had contained C.tarsalis pupae, exuviae and emerging adults(emergence water) . Each sample consisted of50 ml of distilled water which had containedabout 300 pupae. The samples were frozen andthen thawed before being put into a cage for twosuccessive nights. All rafts were laid on thesecond night, thirty-nine in the emergencewater and twenty-one in the pupal water.

In XIb, pupal and emergence waters werecollected, evaporated and freeze-dried. Samplescontaining 16 .5 mg each of the two materialswere reconstituted, each in 50 ml of distilledwater and put into a cage for two successivenights. Eleven rafts were laid, five in the pupalwater and six in the emergence water .

From both XI a and b it appears that bothpupal and emergence water stimulate ovipositionbut that the females showed some preferencefor emergence water under the conditions givenin (a) above .

It was of additional interest to find out whetherthe selection shown by C . tarsalis females wasshown by other species. Culex pipiens femaleswere offered a choice between C. pipiens emerg-ence water and C. tarsalis emergence water .

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The experiment was run for 34 days, accordingto procedure A. Five hundred and ninety-sevenrafts were laid in the C. pipiens dish and 387in the C. tarsalis dish and a preference was in-dicated in 30 out of 34 tests. From these resultsit appears that C. pipiens females also preferwater which has contained developing stagesof the same species, although they may be lessdiscriminating than C. tarsalis females. A brieftest with A . aegypti mosquitoes indicated thatthese females did not discriminate between A .aegypti and C. tarsalis emergence waters .

DiscussionThe preference shown by gravid Culex tarsalis

females for water which has contained develop-ing stages of this species appears to be a responseprimarily to a non-volatile material present inthe water rather than to odour emanating fromit. This conclusion is based on the evidencethat boiled samples of emergence water receivedlarger numbers of rafts than unboiled controlsamples and that distillates obtained fromemergence water received very few rafts whencompared with residual matter .

A non-volatile factor such as is indicated bythese results exerts its effects as the insect makescontact with the water. This permits a consider-ation of three types of chemical stimuli as de-fined by Dethier et al., 1960 ; an arrestant whichmay stop or slow the insect as it contacts thematerial, an ovipositional stimulant whichelicits oviposition by the insect or an ovi-position deterrent which inhibits oviposition .The criteria used to determine the effectivenessof the C. tarsalis factor were the difference in thenumber of eggs laid in the factor-containingwater and a control or contrasting solutionand also the number of times the factor-con-taining dish received more rafts than thecontrol. These criteria indicated that emergence,pupal and larval water all provided an ovi-positional stimulant, but there is no clearevidence that an arrestant was present nor thatthe control dish contained a deterrent . Support-ing evidence for the absence of an arrestantwas obtained from observations made duringthe pre-oviposition period which occurs atsunset for C. tarsalis mosquitoes. As the lightdecreased the insects became increasingly activeand frequently flew across the laying dishes,sometimes landing on the surface but neverremaining there. At this time there was noevidence of a preference . If the factor underconsideration had been acting as an arrestant

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it seems probable that the females would haveremained on the surface at first contact. How-ever, since they did not do so, it seems likelythat the advent of darkness played a part in therelease of oviposition behaviour and thatfollowing this the females became responsive tothe ovipositional stimulant .

The response to a non-volatile ovipositionalstimulant must be initiated by the contactchemoreceptors on the legs and mouthparts ofthe female and there seems little doubt thatchemoreceptors are to be found on the tarsi ofmosquitoes as they are on the legs, mouth partsand ovipositors of other insects (see Dethier &Chadwick, 1948 and Dethier, 1963, for generalreviews ; also Wallis, 1954 ; Hudson, 1956 ; Feiret al., 1961 ; Slifer, 1962). Also the recent workof Ikeshoji (1966) has suggested that the pro-boscis, antennae and tarsi of Culex fatigansare responsible for the preference shown forwater obtained from the breeding sites of thespecies .

By analogy with the work of Evans & Dethier,1957 and Hudson 1958, in which the thresholdof the contact chemoreceptors of Phormiaregina Meigen was lowered by periods of flight,the threshold of the contact chemoreceptorsof mosquitoes may be lowered by flight duringthe period prior to oviposition . Thus, undernatural conditions a female which had flowna long way would respond readily to a lowconcentration of ovipositional stimulant, but afemale which had flown a short distance wouldrequire additional time before the thresholddropped to a level at which the stimulus couldbe detected . This hypothesis is supported by theobservation that in caged colonies egg-layingwas delayed, often beyond the median time foregg development.

It is extremely difficult to determine the sig-nificance of such an ovipositional stimulant inthe choice of a natural oviposition site, particu-larly on consideration of the wide variety ofhabitats in which C. tarsalis larvae are found .These may be either temporary or permanentwater ranging from filthy pools in cattle yards(Hearle, 1926) to the relatively clear water oflake shores (Rueger et al., 1964) .

It is possible that the selection of highlypolluted pools may have some relationship tothe proximity of the host animals ; thus, whenfeeding and oviposition sites are close togetherthere is no opportunity for decreasing thethreshold by expending energy in flight and onlyhigh concentrations of oviposition stimulants

ANIMAL BEHAVIOUR, 15, 2-3

elicit a response ; such concentrations may beavailable in pools of small volume . Conversely,in more sparsely populated areas greater dis-tances between the hosts and the ovipositionsites may promote egg-laying in water contain-ing lower concentrations of an ovipositionalstimulant .The discrimination shown by C. tarsalis

females between water which has containeddeveloping stages of C. tarsalis and water whichhas contained a different species is perhaps themost significant finding in this work, since itsuggests that the female is able to detect abreeding site which has successfully providedthe requirements for development of her ownspecies . There is also evidence that this is truefor Culex pipiens females, although apparentlynot for Aedes aegypti.

Summary1 . Culex tarsalis females selected water con-

taining pupae, exuviae and emerging adults oftheir own species for oviposition in preferenceto distilled water .

2. Preference was shown for water which hadcontained these stages (emergence water) evenafter they had been removed by filtration . Whenthis water was boiled and offered to C . tarsalisfemales with an unboiled sample of the samewater, preference was shown for the boiledmaterial .

3. Evaporation of a volume of emergencewater was followed by reconstitution of the drymaterial with distilled water or with 0 .03MNaCl solution . The reconstituted samples wereselected by ovipositing C . tarsalis females whenoffered together with distilled water or 0 .03MNaCl.4. Ovipositing C. tarsalis females selected

emergence water which had contained the samespecies in preference to emergence water ob-tained from Culiseta inornata, Aedes aegyptiand Culex pipiens.

5. Emergence waters which contained C .tarsalis males and females respectively wereequally acceptable to C. tarsalis females.

6. No discrimination was made by ovipositingfemales between water which had contained C .tarsalis 4th-instar larvae and emergence waterof the species .7. No clear discrimination was made by ovi-

positing females between emergence water andwater which had contained only pupae of thespecies .

HUDSON & McLINTOCK: CHEMICAL FACTOR IN OVIPOSITION

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8. A similar type of behaviour was demon-strated by Culex pipiens but not by Aedes aegyptifemales .

REFERENCESBates, M. (1941) . Studies in the technique of raising

anopheline larvae . Am. J. trop. Med., 21, 103-122 .Bates, M. (1949) . The Natural History of Mosquitoes .

New York: Macmillan .Buxton, P. A. & Hopkins, G . H. E. (1927) . Researches

in Polynesia and Melanesia . London School ofHygiene and Tropical Medicine .

Dethier, V. G. (1963). The Physiology of Insect Senses .London : Methuen .

Dethier, V . G ., Barton Browne, L. & Smith, Carroll N .(1960). The designation of chemicals in terms ofthe responses they elicit from insects . J. econ .Ent., 53, 134-136.

Dethier, V. G. & Chadwick, L. E. (1948) . Chemo-reception in insects . Physiol. Rev., 28, 220-254.

Detinova, T. S . (1936) . 'Certains 616ments de la conduitedes femelles d'Anopheles maculipennis messeae'(in Russian). Medskaya Parazit, 5, 525-543 .(Rev . appl. Ent. B, 25, 141) .

Evans, D. R . & Detheir, V. G. (1957). The regulation oftaste thresholds for sugars in the blowfly . J. InsectPhysiol., 1, 3-17.

Feir, Dorothy, Lengy, J . I. & Owen, W. G. (1961).Contact chemoreception in the mosquito, Culisetainornata (Williston) ; sensitivity of the tarsi andlabella to sucrose and glucose . J. Insect . Physiol.,6, 13-20.

Gjullin, C. M., Johnsen, J. O . & Plapp, F. W ., Jr . (1965).The effect of odors released by various waters onthe oviposition sites of two species of Culex .Mosquito News, 25, 268-271 .

Hudson, B. N. A. (1956) . The behaviour of the femalemosquito in selecting water for oviposition . J.exp . Biol., 33, 478-492 .

Hudson, Anne (1958). The effect of flight on the tastethreshold and carbohydrate utilization of Phormiaregina (Meigen) . J. Insect. Physiol., 1, 293-304 .

Ikeshoji, T. (1966). Studies on mosquito attractants andstimulants . Part III. The presence in mosquitobreeding waters of a factor which stimulatesoviposition. Jap . J. exp. Med., 36, 67-72.

Muirhead-Thomson, R. C. (1951) . Mosquito Behaviourin Relation to Malaria Transmission and Controlin the Tropics. London : Arnold .

Rueger, M . E ., Price, R . D. & Olson, T . A. (1964).Larval habitats of Culex tarsalis Coq. (Diptera :Culicidae) in Minnesota . Mosquito News, 24,39-42.

Russell, P. F. & Rao, T. R. (1942). On relation ofmechanical obstruction and shade to ovipositingof Anopheles culicifacies . J. exp. Zool., 91, 303-329 .

Siegel, S . (1956) . Nonparametric Statistics for the Be-havioural Sciences . New York : McGraw-Hill .

Slifer, E . H. (1962) . Sensory hairs with permeable tipson the tarsi of the yellow-fever mosquito, Aedesaegypti. Ann . ent. Soc . Am ., 55, 531-535 .

Wallis, R. C. (1954) . A study of oviposition behaviourof mosquitoes . Am . J. Hyg ., 60, 135-168 .

(Received 17 November 1966 ; revised 23 January 1967 ;Ms. number : 690)