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A periodical record of entomological investigations, published at the Department of Entomology, Uni- versity of Alberta, Edmonton, Canada

VOLUME I

1965

CONTENTS

Editorial- Words, words, words i

Khan - Behaviour of Aedes mosquitoes in relation

to repellents 1

Book review 36

Editorial- Beastly teachers 39

Pucat - The functional morphology of the mouthparts

of some mosquito larvae 41

Freitag - A revision of the North American species of the Cicindela maritima group with a study of hybridization

between Cicindela duodecimguttata and oregona 87

Guest editorial- Two cultures and the information explosion . . * 171

Wellington - An approach to a problem in

population dynamics 175

Wada - Population studies on Edmonton mosquitoes 187

Wada - Effect of larval density on the development

of Aedes aegypti (L. ) and the size of adults 223

Announcement 250

Corrigenda 250

INDEX

Aedes , 1,41, 187,223 aegypti , 1, 46, 64, 71

campestris > 198

canadensis , 58, 61, 69> 78, 81, 198

cantator » 29

cataphylla ) 5, 69, 71, 195 communis , 69, 73, 188, 195

dorsalis } 198

excrucians f 61,71,78,195,197

fasciata , 51

fitchi , 46,50,54,61,63,76, 78, 195

hexodontus , 59, 61, 78, 188, 195

impiger , 61

implicatus , 61, 195 increpitus , 61, 195, 197 intrudens 5, 188, 195 niphadopsis , 189 pionips , 61, 78 pullatus , 189

punctor , 5,61,71, 188, 195 riparius , 61,69, 195, 197

sollicitans » 29

Stic tic us t 61,69, 195, 197 stimulans » 61,78, 195, 197 vexons* 49,61,78 Amoore, J.E., 4,31 Anabaena , 7 6 Anderson, E. , 89, 167 Andrewartha, H. G. , 201,221 Ankistrodesmus , 76 Anopheles , 44, 72, 75, 81, 194 earlei , 72, 81 fasciatus , 44 gambiae » 223

maculipennist 24, 29, 42, 62, 80 messeae » 79

quadrimaculatus » 42, 49, 60, 190 rossi > 44 Anophelinae, 43 anopheline larvae, 80, 249 Anthon, H. , 49, 82 Anscombe, F. J. , 201,221 Apoidea, 163 Applegarth, A. G. , 56, 82 Apterobittacus , 56, 58 Asaphidion , 36

attractant, 4,21,28 Atwood, C.E., 3,34 autogeny, 223 Baker, F. C. , 219, 221 Ball, G. E. , 38 Banks, C.S., 13,31 Bates, M. , 5, 31 barrier, communication, 172 geographic, 115, 133, 165, 166 Bar-Zeev, M. , 18,31 Beckel, W. E. , 188, 221 bees, dancing, 180 honey, 120, 164 behavior , blood feeding, 1,2 feeding, 41, 72, 73 group, 66 individual, 178 mosquito, 1 orthokinetic, 72 variations, 179 Bekker, E. , 42, 82 Bembidion , 36

graciliforme > 37

humboldtienset 37 'Kl

immaturum » J '

mcrematum » J 1 nigrum f 37

Bibio » 56, 58

binomial distribution, 192,201,

208

Bishop, A. , 2, 31 Blackwelder, E. , 133, 167 Blatchley, W. S. , 103,167 Bliss, C.I. , 201,221 Bock, J. W. , 163, 167 Bowman, M.C., 28,32 Brown, A. W. A. , 4, 31 Brown, W. L. , 90, 167 Browne, B. L. , 15, 31 browser, 42,58,65,74,80,81 Burgess, L. , 28,31 Butt, F. H. , 49, 82 Cain, A. J. , 160, 167

Calliphora erythrocephalat 80 cannibalism, 73, 74 Carabidae, 36, 120 Carabus , 120

Carmichael, A. G. , 29,31

Carpenter, S. J. , 64, 82 Carr, F.S. , 152, 168 Casey, T. L. , 36 Chadwick, L. E. , 27,32 Chaoboridae, 43 Chaoborus , 43, 73, 75, 80

americanus , 41,63,73,75,78 chemor eceptor s, 1, 3, 8, 10, 15 Chir onomidae, 49 Chironomus , 46, 51, 60 hyperboreus , 80

Chitty, D., 177,185 Chlamydomonas , 77 Christophers, S.R., 3,31,42

Cicindela ,audax , 111

bellissima , 87, 94, 101

bucolica , 102

californica , 91 columbica , 87, 94, 101 depressula , 87, 92, 101, 138 duodecimguttata , 87, 91> 101,144

guttifera » 111, 126

hirticollis » 87 > 94, 101, 161

hudsonicat 102

limbata . 87,94,101,161 oregona > 87,92,101,111,144

ovalipennis ,111 praetextata ,91 prove ns is , 111

quadripennis , 1 1 1

repanda , 94, 103

s cute llaris » 111,126

sterope , 1 1 1

tranquebarica , 126

theatina » 87 ,97, 102 Cladosporium , 76 Clements, A.N., 42,82 corrigenda, 250 Coggeshall, A. S. , 80 Cohn, G. , 27, 31 color, elytra, 122, 139, 140, pattern, 88,113,120,122,144 Compositae , 77 Contia tenuis , 143 Cook, E. F. , 42, 82 Corvus corone , 159 Cox, E. L. , 209, 222 Cryophila lapponica , 74 Cu/e*, 41, 44, 58, 74, 80, 194 annulatus , 44

Culex( cont. ) atratus , 44 fatigans , 44 molestus , 24,49,223 nemorosus , 44 peccator , 44 pipiens , 29,44,61,223

tarsalis , 6 1

territans , 51,54,58,61,72,75

Culicoides circumscriptus , 80

Culicidae, 58 Culicinae, 43

Culiseta f 41, 44, 58, 74, 80, 194 impatiens , 58, 61, 69, 78, 81 incidens , 55, 60, 78 inomata , 46,55,58, 63,66,71 , 73,75,77,79,80,81 morsitans , 51, 54, 58, 61, 63, 66, 71, 73, 75, 77, 79, 187 current feeding, 41,68,79 Cyclops , 68, 77 Daphnia , 68

Das, G. M. , 56, 82 Davidson, R . H. , 4,32 Davies, J. T. , 4, 31 DeLong,D.M., 2,31 Dethier, V. G. , 2,32 diapause, 219

Dicaelus , 12 0

Diptera, 56, 58, 62 Dobzhansky, T. , 158, 168 DuPorte, E.M., 50,82 Dyar, H. G. , 42, 83 Dyson, G.M., 27,32 ecophenotypes , 126 elytral pattern, 88, 103, 105, 109, 112, 120, 146, 160 emergence, 218 Eucorethra , 43, 81 underwoodi , 7 3 Euglena , 77, 80 Evans, D.R., 11,32 evolution, 43, 80, 109, 133,163 Ferris, G.F. , 49,83 filter feeders, 42,58,72,74, 79 Findley, J.S., 143,168 Fisher, R. A. , 204, 221 flight, 220 food, 190, 192

shortage of, 224, 228

Foskett, D. J. , 172,174 Fowler, H. W. , 219, 221 Fraenkel, G.S., 72,83 Fragilaria , 7 6 Fr eitag, R . , 87 Frings, H. , 3, 32 Frisch, K. von, 180,185 Geminella , 7 6 genes, 158

infiltration, 150 pleiotropic, 146 geneticist, 173 genitalia, 88, 91, 102, 161 geologist, 172 Gilchrist, B.M., 2,31 Gillies, M. T. , 223, 249 Gleocapsa , 76 Goeldi, E.A., 5,32 Gomphonema , 7 6 Gordon, R.M., 5,32 Gouck, H.K. , 28,32 Gouin, F. J. , 60, 83 Graves, R.C., 103,168 Gressitt, J.S., 165,168 Gryllus luctuosus , 49 Gunn, D. L. , 72, 83 Gunther, A., ii Haddow, A. J. , 5, 32 Hagen, H.} ii

Hamilton, C.C., 87,168 Hammond, A.R., 60,84 Hamrum, C. L. , 3, 32 Hanson, N.R . , 183, 185 Harrison, G.A., 160,167 Hatch, M. H. , 111, 168 Haufe, W.O. , 190,221 Hayward,R., 36 Henry, L. M. , 49, 83 Hinton, E. H. , 56,83 Hocking, B. , ii, 19, 40, 32 Hodgson, E.S., 29,32 homodynamy, 163 Hooke, R . , 41,83 Horn, W. , i

Horsfall, W.R. , 190,221 Howard, L. O. , 42, 83 Howland, L. J. , 80, 83 Howlett, F.M., 4,33 Hoyle, F., 172,174 Hubbell, T.H., 120,168

Hubbs, C. L. , 133, 168 hybrid index, 87, 89, 105, 144* 146 zone, 89, 90, 144, 160 hybridization, 87, 144, 150, 152, 158, 163, 166 Imms, A.D., 56,84 Inger, R . F. , 90, 169 intergradation, 87, 90, 103, 134, 144, 148, 159 introgression, 126, 152, 162 isolation, 159, 166 differentiation, 143 geographical, 164 spatial, 134 James, H. G. , 74> 84 Johannsen, O.A. , 42,84 Johnston, J. W. , 4, 31 Jones, F. N. , 4,33 Jones, J. C . , 60, 84 Kalmus, H. , 1,33 Kellogg, F.E., 5,33 Kemper, H. , 28, 33 Kendrew, W. G. , 125,169 Kennedy, J.S., 24,33 key, 101 Khan, A. A. , 1 Khelevin, N. V. , 219,222 King, P. B. , 164, 169 Klomp, H. , 224, 249 Knab, F. , 42,83 Knight, K. L. , 189, 222 Krishnamurthy, B . S. , 223,249 Kupka, H. , 28, 34 LaCasse, W. J. , 64, 82 larvae, mosquito active, 180

browsing, 41,51,62,70,74,77 density, 223 development, 223 filter feeding, 41,42,63,77 labium, 59 labrum, 50 mortality, 223 non- predatory, 41 predatory, 41, 43, 63, 73, 74, 77 overcrowding, 223, 249 sluggish, 180 Laven, H. , 223,249 Leng, C. W. , 87, 169 Lepidoptera, 62, 163

Lindroth, C.H. , 36 Linsley, E. G. , 163, 169 Lotmar , R . , 8,35 Lumsden, W.H.R., 5,32 Lulzia » 43 halifaxi > 50

Macfie, J. W.S., 10,33 Malacosoma pluviale , 175 Manton, S.M., 46 Martin, P. S. , 164 mating, 13,25,27,157 Mayr, E. , 90, 169 MacGinitie, H. D. , 164, 169 McGregor, D. , 80, 84 McLintock, J. , 46, 84 Mecham, J.S., 158,169 mechanor eceptor s, 1,3,29 Mecoptera, 46, 56 Meinert, F. , 42, 84

Melanoplus puer , 120

Mellon, De F. , 29, 32 Menees, J.H., 46,84 Mengel, R.M. , 164, 169 Miall, L. C. , 42, 84

Microspora , 7 6 migration, 220 Miller, R.R. , 133, 168 Miocene, 164 Mitchell, E., 42,84 Mochlonyx , 43, 75, 80, 81

culiciformis , 74

'velulinus > 41,63,73,74,78 Montchadsky, A.S., 42,84 Morita, H. , 30, 33 morphology, 91 Morris, R.F. , 204,222 mortality, 187,189,221,223, 225,235

mosquitoes, black-legged, 187 control of, 187,217 Edmonton, 187 mouthparts, 41,42,64,81 mutation, 109, 163 Navicula , 76 Nearctic, 165 Nematocera, 46, 56, 58, 62 Nuttall, G.H.F., 42,85 Ochlerotatus , 187, 196, 197 Olbiogaster , 49 olfaction, 4, 27

Omus californicus Oncopeltus fasciatus , 49 Opifex fuscus , 80

overwintering, 220 oviposition, 15, 17, 25, 27, 30,

190, 194, 219, 221 Owen, A. R . G. , 202,221 Palearctic, 165 Panorpa , 56, 58, 62 Panorpoidea, 58 Papp, H. , 91, 169 Peffly, R. L. , 4, 32 Peters, W. , 8, 33 Peterson, A. , 43, 85 Phacus , 77 phenology, 139 Phormia regina , 11,15,29

Phrypeus > 36 phylogeny, 160 P innularia , 7 6 Pinus , 77 Platt, J.R., 177 Pleistocene, 133, 144, 158, 163 Pliocene, 165, 166 Poisson distribution, 200, 203 population, allopatric, 158 alpine, 122, 125 boreal, 122

density, 190,192,201,210, 216 desert, 125

dynamic s, 175,221,223 ecology, 177, 184 literature, 176 primitive, 166 samples, 89, 121 studies, 187 theory, 177, 179, 184 world, 171,172 Populus , 77 Potter, E. , 56, 85 predators, 43,73,74,81 Provost, M. W. , 220, 222 Pucat, A.M., 41 pupation, 224, 228 Puri, I. M. , 42, 85 Putnam, P. , 14, 34 Quate, L. W. , 163, 169

Quiscalus quiscula 159

Rahm, U. , 3, 33

R ana aurora * ^43

Rao, T.R. , 24, 34 Raschke, E. W. , 42, 85 Rausch, R . L. , 120,169 Reaumur, M. , 41,85 receptors, 1

olfactory, 4, 16, 30 R eed, W. , 1 Rees, B. E. , 56, 83 Rempel, J. G. , 64,85 Renn, C. E. , 42,85 repellents, 1 R euter , J. , 4, 34 Richards, D. W. , 14,34 Ridgway, R., 88,169 Rivalier, E. , 91, 169 Roeder, K.D. , 29, 33 R oss, R . , 1, 34 Roth, L.M. , 3, 34 Rubin, M . , 4,31 Rumpp, N. L. , 91,170 Sabrosky, C. W. , 163,170 Salem, H. H. , 51, 85 Saltatoria, 164 Sass, J. E. , 75,85 Scenedesmus, 76 Schenkling, K. , i Schremmer, F. , 43, 85 Sekhon, S.S., 3,34 sex hybrid, 90 Shaerffenber g, B. , 28, 34 Shalaby, A.M., 46,85 Shannon, R . C. , 14, 34 Shelford, V. E. , 122,170 Shipley, A.E., 42,85 Short, L. L. , 89, 170 Shute, G. T. , 223, 249 Sibley, C. G. , 89, 170 Simulium , 7 9 Slifer, E.H. , 3, 34 Smith, C.N., 29,35 Snodgrass, R.E., 42,86 Snow, C . P. , 172 Sorex vagrans , 143 Southwood, T.R.E., 220,222 Spielman, A., 223,249 Spiro gyra , 76, 77 Stace-Smith, G. , 148 Stagmomantis Carolina , 49

Stahler , N. , 223,249

Stauroneis f 76

Stebbins, R.C., 143,170 Steward, C. C. , 3,34 Sturckow, B. , 30, 34 Sturtevant, A. H. , 163,170 subspecies, 125,434,139,143 sugar feeding, 10,25,27, 30 Sullivan, C.R., 177,185 Surtees, G. , 42, 86 Sylvester-Bradley, P.C.,163,170 Sylvester, E. S. , 209,222 synonymy, 103, 112, 139 Systematic Zoology, 90 taxonomist, 173 taxonomy, 97, 125 teachers, 39 Telford, A. D. , 219, 222 temperature, 190, 192, 219, 233 Tertiary, 163,164

Theobaldia incidensJS5 , 60 thermoreceptors, 1,2,30 Thiel, Van, P.H. , 4, 34 Tipula , 56, 58 Travis, B. V. , 29, 35 Trechus , 36

Trembley, H. L. , 46, 86 Ulmus , 7 1

variation, color , 105, 106,112,141 geographic, 89, 103, 112, 134 interspecific, 88, 91, 97 intraspecific, 88 population, 109, 112 Venard, C.E., 4,32 Vimmer, A. , 49, 86 Vockeroth, J.R., 188,222 Wada, Y., 187,223 Wallis, J. B. , 4, 35 Waters, W.E., 201,222 Weismann, R., 8,35 Wellington, W. G. , 175,185 Wesenberg- Lund, C.N., 42,86 Wheeler, W.M., 49,86 Williams, T.R., 80,86 Willis, E. R. , 3, 35 Wilson, E. O. , 90, 170 Winteringham, F.P.W., 174 Wright, R. H. , 5, 35 Xiphidium ensiferum , 49 Yost, M. T. , 8, 32 Zeuner, F. E. , 163,170 zoogeography, 160, 163

Quaestiones

entomologicae

A periodical record of entomological investigations, published at the Department of Entomology, Uni- versity of Alberta, Edmonton, Canada.

VOLUME I

NUMBER 1

JANUARY 1965

QUAESTIONES ENTOMOLOGICAE

A periodical record of entomological investigations, published at the Department of Entomology, University of Alberta, Edmonton, Alberta.

Volume 1 Number 1 2 January 1965

CONTENTS

Editorial i

Khan - Behaviour of Aedes mosquitoes in relation

to repellents 1

Book review 36

Editorial - Words, words, words

The first edition of the World List of Scientific Periodicals, published in 1921, listed 25,000 titles. The second edition in 1934 listed more than 36,000; the third edition in 1952 listed more than 50, 000. The fourth edition now appearing lists over 60, 000, despite the fact that "some 10, 000 titles included in the third edition have been left out as being of social or commercial rather than scientific inter est". Most periodicals have recently waxed fat, so that one may estimate 25 years as the time in which the flow of scientific literature doubles itself.

By comparison with science as a whole, the growth of entomo- logical literature seems somewhat pedestrian; the Insecta portion of the Zoological Record listed 1970 titles of papers in 1921 and 4024 in 1953. The applied literature, as represented by the Review of Applied Entomology has been, somewhat surprisingly, growing more slowly than this, so that one may estimate 35 years for the entomological lit- erature to double itself. Even so the 25,229 entomological articles listed in Horn and Schenkling as published from the beginning of history until the end of 1863, at current rates would be produced in about four years, and the total number of scientific papers now published in the field of entomology must exceed a quarter of a million. One may sus- pect, however, a shrinkage in the mean length of papers under the joint influence of mounting page charges and the philosophy of "publish or perish" coupled with the waning ability of administrators to judge publications by anything beyond their number.

Some may say that in this situation a new periodical should be offered with an apology - if at all. But if we would slow down the march of science, we must stop research before it has begun, not lose the results of it when it is all but finished. Certainly we must see to it

11

that we do not produce new facts faster than we can assimilate them into generalizations, although this process calls for that very breadth of outlook which the literature flood makes it difficult for us to achieve. If we can no longer achieve individual breadth, we must provide for composite breadth by facilitating diversity of training and the unusual combination of subjects.

If we stagger under the impact of a swelling literature, before we call for a slow down in research we should remind ourselves that a quarter of a million entomological papers only represents less than one per described species of beetle, and that more than half the species of insects remain to be found and described.

If then, this growth of the literature must go on, what can we do to keep abreast of it? A great many things: fight the trend to shorter

papers, which has now reached the ridiculous stage when an index card for a paper may be larger than the content of the paper itself. It costs more in time, money, and effort, to produce, file, store, retrieve, and read ten one page papers than one ten page paper. Publish in the most appropriate periodical from the subject viewpoint; publish promptly; index and abstract everything diversely; and make full use of modern techniques such as microforms, punch cards, and even computers. It may seem redundant to say that material should be published once only, yet how often do we find it difficult to avoid duplicate publication of material from the proceedings of a meeting, and how often is this due to inappropriate publication in the first place? A marriage between microcards and punch cards is long overdue; if sufficiently prolific, the hybrid offspring would be of inestimable value to the bibliogr? pher .

There are signs that things are beginning to move in this direc- tion; perhaps this periodical is one of them. But one may question whether the move is fast enough to get us out of chaos: movable type ,

despite its name, is conservative stuff.

Despite our concern for the future, we should be both remiss and churlish to enter 1965 without a backward glance to 1865 and the beginning of the Zoological R ecord. Let us pay both dollars and respect to our venerable abstracting and indexing services - in no other field of endeavour is continuity more important. I wonder whether any other branch of science is as fortunate as entomology with its Hagen, Horn and Schenkling, and Zoological Record. Many complain of the increasing delay in publication of successive volumes of Zoological Record, but how many of the complainants have ever attempted a similar task? And whose fault is this? As Glinther pointed out in his preface to vol- ume one in August 1865, many journals of learned societies which would carry the date 1864 on their title pages, had still not appeared; but here we are treading on dangerous ground. We regard it as a most fortunate and propitious honour, to commence publication in the year in which the Zoological Record celebrates its centenary.

Brian Hocking

EFFECTS OF REPELLENTS ON MOSQUITO BEHAVIOR

Department o t Entomology Qvaestiones entomologicae

University of Alberta i.i-jo. ivo d

The behavior of Aedes aegypti L- and other species of Aedes in relation to repellent chemicals was studied. The repellents used were dimethyl phthalate, ethyl hexaned- iol, N, N-diethyl metatoluamide and indalone. The effect of these repellents on the behaviour of mosquitoes was studied firstly by placing the repellents on selected parts of the environment and secondly by painting them on parts of mosquitoes themselves where chemoreceptors are known to occur, such as the antennae, labium, and tarsi. The aspects of behavior studied were: feeding on blood and on sugars, mating, oviposition, the reactions to wind, geotaxis and orient- ation to centrifugal force, and the visual response to black stripes. All these aspects of behav- ior are affected significantly by repellents. Dimethyl phthalate has the greatest effect of the four repellents on blood feeding behavior when they are painted on the tarsal receptors and the smallest effect when they are painted on the receptors of the antennae and the labium.

The experiments provided some understanding of the mode of action of insect repel- lents. They suggest that repellents interfere with normal behavior perhaps by blocking the olfactory receptors mediating attraction to food and the contact chemoreceptors invoking feeding on blood and those used in the selection of oviposition sites. The experiments show that mech- anoreceptors effecting orientation to gravity and air flow and visual receptors effecting orientat- ion to black stripes are also interfered with by repellents. There is also some evidence that repellents block the thermoreceptors which may mediate piercing for feeding on blood and perhaps auditory organs involved in mating. The only receptors which the repellents do not appear to interfere with seem to be those of the common chemical sense.

INTRODUCTION

The discovery of the transmis sion of malarial parasites by Ross (1898) and the discovery by Walter Reed and his collaborators that yellow fever was transmitted by Aedes aegypti led to the realization of the importance of mosquitoes as carrier of disease. Repellents being a cheap and efficient means of individual protection, many worker s studied their effects mainly against the blood feeding behavior of insects. Kalmus Hocking (I960), however, studied some other aspects of behavior as well. I have studied the behavior of Aedes aegypti in the presence of repellents not only in relation to blood feeding but also in relation to sugar feeding, mating, oviposition, geotaxis, wind direction and speeds, and visual responses to black stripes. The repellents used were: dimethyl phthalate, indalone, diethyl toluamide, and Rutger's 612. The first two are esters, the third an amide and the last named an alcohol. These are compounds of low volatility and moderate molecular weight. They are insoluble or only very slightly soluble in water but are miscible with alcohol and ether. Their physical and chemical properties are listed in table 1.

z

Repellent Effects

TABLE 1 - Chemical and physical properties of the repellents used in the study of behavior oi Aedes .

Common Name	Chemical Name	Mol. Wt.	Boiling Point	Solubility and Miscibility
Dimethyl phthalate	dimethyl benzene- ortho -dicar boxylate	194. 18	285°C	0. 43% w/w soluble in water
Indalone	n-butyl mesityl- oxide oxalate	226. 26	1 1 3°C	Insoluble in water; miscible with alcohol
Diethyl toluamide	N, N- diethyl m-toluamide	191	111°C at 1mm	Insoluble in water; miscible with alcohol
Rutger *s 612	2 -ethyl, 1, 3- hexanediol	146. 22	244°C	Slightly soluble in water; mis- cible with alcohol

Blood feeding behavior was studied by applying the repellents in various ways in the environment and on different chemosensory fields of female Aedes aegypti. Some general observations were also made on the blood feeding behavior of Aedes spp. mosquitoes in the field and Aedes aegypti in the laboratory. A preliminary test was made of the effect of washing chemosensory areas with lipoid solvents on blood feeding by Aedes aegypti.

REVIEW

Sense Organs of Aedes aegypti L.

An Aedes aegypti female is attracted to its host in part through the chemoreceptors located on head appendages, mainly the antennae. Bishop and Gilchrist (1946) showed that in Aedes aegypti eyes are not essential for feeding on blood. Roth (1951, p. 60) also reported that eyes are not necessary in locating the host in a small cage.

DeLong (1946) considered the anteranae and the palps as the chief organs for locating the host and stimulating probing. According to him the antennae may perform both functions but the palps can receive stimuli only when the insect is directly on the skin. Roth (1951) considered that the antennae function as directional thermoreceptor s and probably chemoreceptors as well. R oth ( 1951 ) also reported temperature receptors on the palps of A. aegypti. Dethier (1952) considered that

Khan

3

different receptor fields function at different levels of sensitivity. The antennae according to him are the most sensitive and the various mouth- parts less so. Rahm (1958) showed by antennal amputation that these organs are essential for host finding and attraction from a distance. He also reported that antenna-less mosquitoes can probe and suck if the palps remain intact.

Antennae

The antennae in the male and female consist of a basal ring-like scape, aglobular pedicel, and a long flagellum of thirteen articles. The pedicel inboth sexes contains Johnston’s organ, which is more developed in the male.

Roth and Willis (1952) reported that many thin walled trichoid sensilla are present on each of the thirteen flagellar articles of the female A. aegypti and on the two terminal flagellar articles of the male. They concluded on experimental evidence that they serve as hygro- receptor s .

Christophers (I960, p. 663) described the trichoid sensilla as . .40-50 p, in length, thin walled and without articulated base, arising from thin membrane over a pore canal surrounded distally by a semi- circular ridge in the article. ”

Steward and Atwood (1963) identified five structural types of sensilla on the antenna of the female A. aegypti. Three of these types they found thin walled and classified them as Al, A2 and A3. According to them a typical Al sensillum is 0. 06 mm long, curved and tapering to a sharp point. Type A2 is shorter, 0. 04 mm long and with a blunt tip. Both are about the same diameter. The innervation of the two types is essentially the same. Steward and Atwood described type A3 as a short, curved, thin-walled peg organ which is innervated by a group of nerve cells. Sensilla of type Al and A2 are more numerous on the distal articles while sensilla of type A3 are found to be located chiefly on the proximal articles of the antennal flagellum. They concluded from experimental evidence that type Al and perhaps A3 play a major role in mediating attraction while type A2 are responsible for mediating repulsion.

Slifer and Sekhon (1962) studied the structur e of the sense organs in the flagellum of A. aegypti. The heavy walled hairs according to them ar e mechanor eceptor s . The thin walled hair s with sharp tips they thought to be chemor eceptor s . The thin- walled hairs with blunt tips they supposed to be olfactory in function.

Palpi

Roth and Willis (1952) described the palps of female Aedes aegypti as abundantly supplied with thin- walled club-shaped sensilla on the terminal segment. Pointed trichoid sensilla are also present. There is also a central short sclerotized peg at the tip of the palp.

Labium

Frings and Hamrum (1950) noted four kinds of hairs on both sexes of A. aegypti. Of these, hairs about 40 \l long and lying at the tip of

4

Repellent Effects

the labella are considered to be tactile in function while curved hairs about 20 pin length at the tip and on the ventral surface are believed to be chemor eceptor s .

Tarsi

On the tarsi of the fore and mid legs of A. aegypti are many slightly curved hairs probably tactile in function (Frings and Hamrum, 1950). Wallis (1954) found that in A . aegypti all tarsal segments were provided with thin-walled curved spines. Slifer (1962) described the hairs on the tarsias approximately 100 in number in the female. These hairs stain at the tip when dye is applied to the external surface of the insect. She concluded: "Little doubt now remains that the hairs with

stainable tips are the tarsal gustatory receptors of the mosquito. "

Mode of Action of Olfactory Receptors

Several theories have been advanced to explain the mode of action of olfactory receptors. Jones and Jones (1953) reviewed the modern theories on olfaction and classified them as; mechanical, chemical, steric, radiation and vibration theories.

Davies (1962) proposed that the mechanism of olfaction is the penetration and dislocation of a small region of the wall of an olfactory nerve cell. This dislocation allows the potassium and sodium ions to move across the membrane, so initiating the nerve impulse.

Amoore (1963), and Amoore, Johnston and Rubin (1964) favor the stereochemical theory of olfaction. According to them the odor of a chemical is determined by the structure of the molecule, in particular by its size and shape. If a chemical is volatile, and its molecules have the appropriate configurations to fit closely into the receptor site, then a nerve impulse will be initiated, possibly through a mechanism involving disorientation and hence depolarization of the receptor cell membrane.

Factors Attracting Mosquitoes to the Host

The mode of action of repellents cannot be fully studied without an understanding of the factors that attract the insect to the host. Contradictory views can be found in the literature on this point; all workers accept temperature and humidity, as attr actant factors; others consider factors like carbon dioxide and host odor, or only carbon dioxide to be also important in attracting the mosquito to its host.

Howlett (1910) believed temperature to be the chief attr actant and said that the smell of sweat or of blood was not attractive. Reuter (1936) showed that moisture was distinctly attractive to A. aegypti. Van Thiel (1937) assigned the role of attr action chiefly to the physical factors of temperature and humidity and the chemical factor, carbon dioxide. Later Van Thiel (1953) considered that the scent of the host plays an important part in the orientation of the mosquito toward it.

DeLong, Davidson, Peffly and Venard (1945) found moistened warm air more attractive to A. aegypti than warm air. Most of their tests were conducted with olfactometers or inanimate objects. Brown (1958) recognized six factors which guide female mosquitoes to their animal hosts, three of these being air-borne (water vapor, carbon dioxide, and

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5

convective heat) and three visual (movement, contour, and r eflectivity) .

Kellogg and Wright ( 1 957 ) and Wright ( 1 96Z ) considered moisture and carbon dioxide to be the main attractant factor s . Christophers (I960, p. 535) remarked: "The evidence that smell is an important stimulus in the attraction of A. aegypti to feed is not very strong. "

On the other hand, many have said that body odor plays an important role in the attraction of mosquitoes . Goeldi (1905) reported per spiration to be the agent attracting mosquitoes to man. Haddow ( 1 942 ) r eported that an unwashed African child attracts more Anopheles spp. than a clean child. Willis ( 1 947 ) r eported that females of A. aegypti and Anopheles quadrimaculatus Say were attracted by the odor of the human arm. He also found CO^ in con- centrations of 1, 10, or 50 per cent in the air not attractive to females of A. aegypti or Anopheles quadrimaculatus when tested in an olfactometer. Bates (1949) thought smell to be the primary stimulus in guiding the mosquito in its sear ch for food. R ahm ( 1 956 ) r eported that CO2 emitted by the skin did not determine attractiveness and remarked ( 1 957) that human odor and sweat may play a part in the attraction of mosquitoes to the human hand. Again in 1 957 he r eported that per spiration did not seem to attract mosq - uitoes but the odors given out by the host did. Rahm ( 1958) further remark - ed that the olfactory substances of man were found to be alone responsible for greater activity offemale A. aegypti. Dethier (1957) wrote: "Host finding and discrimination, trail following, orientation to odor s by flying insects and courtship are shown to depend largely on the chemical stimuli. "

EXPERIMENTAL - BEHAVIOUR

Blood Feeding in Relation to Repellents

Christophers (I960, p. 486) remarked on blood feeding by a. aegypti in the following words: "Another striking feature of feeding is that the ins- ect once it has begun to suck blood, appears to become oblivious to all dan- ger and considerable physical force is required to make it give up its hold." Thi s featureis referredtoby Gordon and Lumsden (1939 )who wr ote that they were only able to get A. aegypti tofeed on the frog's foot by employing mos - quitoes which had been allowed to start feeding on the human arm. When nearing repletion, however , the insect usually leaves readily if disturbed.

Kalmus and Hocking (I960) observed the effect of painting repellent with a fine camel hair brush on the backs of feeding mosquitoes . A lead was taken from this study andmore observations were made on the effect of re- pellents on other species of Aedes in the field and Aedes aegypti in the labor- atory.

Observations on Aedes spp. in the Field

For studies on the species of Aedes in the field a thicket of poplar trees was selected. The four repellents , dimethylphthalate, ethyl hexan- ediol, indalone, andN-N-diethylmetatoluamide were used. The mosqui- toes reacted to all four repellents in the same way. The species of Aedes studied were A. punctor Kirby, A. cataphylla Dyar, and A. intrudens Dyar.

The time to take a complete blood meal, from the insertion of

Repellent Effects

6

the proboscis to its retraction after complete engorgement ranged from two to four minutes. (Mean = 2 min 31 sec with standard deviation 41 sec). It was observed that the mosquitoes could be very easily disturbed in the early stages of their blood meal. If a clean brush were brought near them soon after the insertion of the proboscis, they could be seen retracting it. If a repellent or olive oil were placed near the antennae or painted on the mesonotum, the mosquitoes invariably flew away. As reported byKalmus and Hocking ( 1 96 0, p. 7 ) "A contact between r epellent chemicals as liquids and substantial areas of the proboscis, tarsi and tibiae, mesonotum or the wings leads to the interruption of biting, and in mosquitoes not engaged in biting to the retraction of the touched limb or limbs or to take off. ^ But the behavior of mosquitoes was found quite different in relation to repellents and other stimuli if they had been feeding for a minute or more, i. e. roughly in the middle of their meal; e. g. :

(i) The mesonotum was rubbed with a dry brush, painted with repellents or olive oil until the whole mesonotum was covered with liquid, but the mosquito never flew away, instead it completed its blood meal, continuing to feed for another 45 seconds tov one minute.

(ii) The antennae were painted with repellents, were in fact soaked in repellent, but the mosquitoes continued to feed.

(iii) A drop of repellent was made to flow near the tarsi, there was no reaction until it made contact with them. As soon as contact was made the tarsus was lifted. The same reaction was observed with olive oil. However, the mosquitoes continued to feed even when the tarsi of all the six legs were lifted. The mosquito then came to rest on its abdomen. When the repellent was presented on a brush near the lifted tarsi, they sometimes rested the tarsi on the repellent soaked brush, without showing any other abnormal behavior, and continued to feed.

(iv) Similar behavior was observed inmosquitoes feeding on the foot through socks. Mosquitoes coming to feed landed only on clean areas of the sock and avoided areas where repellent had been placed. However, mosquitoes which had been feeding through the sock for some time were not affected if a repellent was placed on the sock underneath them, and they continued to feed to completion although they lifted the abdomen.

(v) Chloroform or ether was brought near the abdomen of a feeding mosquito. It always flew away, even when it had been feeding for a minute or more.

( vi ) A hot spatula was brought near the mosquito (about 1 mm). The spatula was heated for two minutes in a flame of a spirit lamp. Eighty per cent of the mosquitoes took off in 5 to 10 seconds. When the spatula heated for the same time was kept at the same distance from the mercury bulb of a Fahrenheit thermometer, the thermometer registered a rise of 4-6 degrees.

(vii) Repellent was painted on the wing of a feeding mosquito. The mosquito always flew away but when the wing was rubbed with a dry brush or painted with olive oil it continued to feed.

(viii) Physical injury was inflicted on the mosquito to the extent

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that all the six legs were clipped off at the femoro - tibial joint, but it continued to feed and did not fly away.

The observations were made at a temperature of 65°F and R.H.

of 57%.

Observations on .Aedes aegypti

In the laboratory the same behavior was studied in Aedes aegypti. A one cubic foot cage made of steel wire and covered with nylon net was fitted with a sleeve on each of two adjacent walls, i. e.at right angles to one another. Mosquitoes were allowed to feed on a hand inserted through one sleeve while the other hand was introduced through the other sleeve to apply the repellent.

As observed in the other species of Aedes, Aedes aegypti couldalso be easily disturbed in the initial stages of blood feeding, but after one minute of feeding they could not be disturbed so easily:

(i) When the mesonotumwas rubbed with a dry brush or painted with olive oil or any of the four repellents under study.

(ii) When their wings were painted with repellents. This was contrary to the behavior observed in the field spe'cies which invariably flew away whenever repellents were painted on the wings.

(iii) They continued to feed even when they were made to rest their tarsi on the repellent soaked brush.

(iv) Being small in size, it was not possible to paint their antennae with repellent while they were feeding, but when a drop of repellent was placed very close to the proboscis they continued to feed.

(v) Almost every mosquito continued to feed when the tarsi of its hind legs were clipped off, but some flew away when the tarsi of their other legs were clipped.

(vi) When a heated spatula was brought near them they always flew away even when the spatula was as far as 1-2 cm away. It had to be brought much near er to mosquitoes in the field to elicit this response. When the spatula heated for the same time was kept at the same distance from the mercury bulb of a Fahrenheit thermometer this registered a rise of 1. 5 to 2 degrees.

Experiments were conducted by applying the repellent on differ ent chemosensory fields of female A. aegypti and observing the behavior and recording the number feeding on an untreated human arm. As the repellent was not applied on the skin, there was no interaction between the skin and the repellent or the chemical stimuli emanating from the skin and the repellent on the surface of the skin. The experiments provided some under standing of the site of action of different repellents as well as providing a quantitative basis for comparing the repellents with each other. The experiments also provided a quantitative basis for evaluating the function and efficiency with which the different chemosensory fields play their role in the act of feeding as well as some grounds for accepting the role of smell in attracting mosquitoes to feed and the function of the repellent when applied on the skin in offsetting this role.

8

Repellent Effects

10- 12 female mosquitoes, 7-8 days old, previously fed on raisins and sugar solution only, in a sucking tube and then chilling them for 1. 5 min at 15°F, in order to immobilize them. Their probosicides , either one or both antennae, or all the tarsi, were then painted with repellents with a fine brush in separate sets of experiments. This operation was performed over a cold petri dish covered with a filter paper and placed under a binocular microscope. A radius was drawn in ink on the filter paper and mosquitoes were treated one by one, starting on one side of the radius until all of them were treated. They were then sucked back into the sucking tube and released in a paper lined petri dish to revive in a one cubic foot cage of steel wire covered with nylon net. The mos - quitoes recovered from the chill in 2-3 minutes. The behavior and the number that fed on blood on introducing the arm into the cage through a sleeve were noted, firstly ten minutes after the treatment and then at greater intervals from the treatment until the number fed in a given time approached the number fed in controls. Two controls were run with each set of experiments, one a plain control when the receptor field that was intended to be treated was rubbed with a vdry brush only, and another when it was painted with olive oil. The palps could not be treated separately without running some repellent on the proboscis and the antennae, because of their close proximity to these structures.

Results - The figures given in table 2 give the cumulative mean percentages of mosquitoes feeding on blood after different chemor eceptor sites were painted with repellents . The standard error of themean was used to find statistical significance between the means.

The results show that Rutger's 612, diethyl toluamide, and indalone reduce the number of mosquitoes feeding on blood more than dimethyl phthalate after the first ten minutes when the proboscis was painted, and the effect lasted longer. Indalone remained significantly more effective as compared to Rutger's 612 and diethyl toluamide after two hours when painted on the proboscis.

When painted on both the antennae, diethyl toluamide, Rutger's 612 and indalone again reduced the number of mosquitoes feeding more than dimethyl phthalate. The effect of dimethyl phthalate was found to have been lost within one hour but the effect of the other three repellents lasted more than six hours.

When painted on one antenna, the same significant differences were found between the repellents as when both the antennae were painted, i. e. , diethyl toluamide, Rutger's 612 and indalone were significantly more effective than dimethyl phthalate.

The results obtained on painting all the tarsi with repellents were, however, different. Dimethyl phthalate was found to reduce feeding more effectively when painted on tarsi than when painted on both the antennae or on the proboscis, and to maintain this effect at least as long as the other three materials.

There is evidence that many repellents work byway of specialized chemor eceptor s (Weismann and Lotmar, 1949; Dethier and Yost, 1952; Peters, 1956; Dethier, 1956 a). Peters (1956) reported that Calliphora erythrocephala could detect dimethyl benzamide with the tarsal

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10

Repellent Effects

receptors only, while other materials like indalone and dimethyl carbate could be detected with the tarsal receptors, labella, and antennae.

The significant difference in the number of mosquitoes landing on the hand after treatment of chemor eceptor s on different head appendages and on the tarsi can be explained on the basis of the population of chemor eceptor s getting such treatment. As most of the chemo- receptors are situated on the antennae, their treatment with repellents would inhibit the landing of mosquitoes on the hand more than the treat- ment of other head appendages. The ineffectiveness of the painting of one antennae only in keeping the mosquitoes from a blood meal for two hours can be explained by the same argument, i. e. , a large population of chemor ec eptor s r emained functioning effectively when only one antenna was painted. The painting of any one of these chemor eceptor sites with repellent must be affecting the mosquito in two ways, affecting the chemor eceptor s of the chemosensory area painted in liquid form and also affecting the adjacent chemosensory sensilla in vapor form. The greater the area painted, the greater the number of sensilla affected, resulting in inhibition of feeding for a longer period.

Painting Repellent on Mosquito Antennae and Host Skin

3y the procedure described above one antenna of each of about 10 A. aegypti females was painted with diethyl toluamide. An arm also treated with diethyl toluamide was then introduced into the cage and the behavior of the mosquitoes was studied. A little more flight activity and some searching on the wing was observed in these mosquitoes as compared to those in the control where no repellent was applied on the mosquitoes themselves but only on the hand. A similar behavior was observed in experiments with the other three repellents as well. In controls, mosquitoes were seen mostly sitting on the walls of the cage. There was little or no flight activity.

When both the antennae of mosquitoes were treated with diethyl toluamide, indalone or Rutger’s 612, and the same repellent was applied on the hand introduced into the cage, the mosquitoes could be seen searching on the wing. Many landed on the repellent coated surface of the hand, walked about and even probed but did not take a blood meal. The behavior was observed for ten minutes every hour for four hours but no mosquito bit. In similar experiments with dimethyl phthalate, however, no landings on the hand were observed though the mosquitoes came quite close to it and sometimes even touched the skin.

Sugar Feeding

The principal food of female Aedes aegypti is blood from a human host though they can exist for long periods on food other than blood . Male Aedes aegypti do not take blood at all but feed entirely on sugary materials. Goeldi (1905) kept females alive for 31 to 102 days on honey alone. Macfie (1915) observed that the females feed on honey for the first couple of days but the males feed only on honey at anytime. Gordon (1922 b) observed both males and females of Aedes aegypti sucking nectar from flowers. Many observers have noted that sugary fluids, raisins, bananas, and other fruits are sucked by both sexes.

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Many workers have devoted much time to studies of the effect of repellents on blood feeding of mosquitoes but their effect on sugar feeding has not attracted much attention. Evans (1961) has studied the effects by the blowfly Phormia regina Meigen. Experiments were conducted to study the effect of repellents on the feeding of Aedes aegypti on raisins.

Kalmus and Hocking (I960) conducted some tests on blood feeding in relation to repellents with Aedes aegypti by keeping a 10 cm length of 3mm outside diameter glass tubing which was clamped in a vertical position so that the lower end was about 1 cm above the middle of a 6 cm bare circle on the back of a gloved hand. A few drops of repellent were placed in the lower end of the tube. In this way a circle of skin about 1. 5 cm diameter was kept free of bites. A lead was taken from this experiment in exploring the effect of repellents on the feeding of Aedes aegypti on raisins.

Experiment 1

About 100 male and 100 female mosquitoes were taken in a cubic foot cage of steel wire covered with nylon net. Thqage of the mosquitoes was 2-4 days and they were not fed anything for six hours prior to experiments. Ten raisins were fixed with 1 cm clear space between each on a horizontal steel wire hanging 4 inches below the top of the cage. The wire was hung by bending its ends and hooking them on top of the side walls of the cage. A 2 cm wide strip of paper was fixed above the raisins, running parallel to them at a distance of 1.5 cm. Half of this paper strip (covering 5 raisins) was painted with repellent and the other half (covering the other 5 raisins) was kept as a control. Observations were made on the number of mosquitoes settling on either side at intervals of 5 minutes. After each observation the cage was shaken and another observation recorded after five minutes. In this way five replicates were taken for each repellent. Separate batches of mosquitoes were taken in separate cages for experiments with different repellents .

The observations are recorded in table 3. The vapor of repel- lents significantly reduced the number of mosquitoes feeding on raisins . The standard error of the mean was used as statistical test for significance.

Experiment 2

Ten raisins were fixed on the wire lying as close to each other as possible without touching. Five alternate raisins were then painted with repellent leaving the other five as controls. The numbers of mosquitoes that settled on the treated and untreated raisins are recorded in table 4, column 1 to 3. The figures are means of 5 replicates. Observations were recorded every five minutes as in the previous experiment. The total number of mosquitoes in the cage for each experiment was 200.

In these experiments mosquitoes were seen coming close to the raisins to land but they usually flew away without landing. No significant difference was found between the number of mosquitoes settling on treated

Repellent Effects

12

and untreated raisins in the control with olive oil. The results show that the repellent on the treated raisins kept the mosquitoes away from the untreated raisins as well. Kalmus and Hocking (I960, p. 23) obtained bites up to almost a mosquito half- width (about 2. 3 mm) from a repellent painted circle on the back of the hand. In these experiments the mean width of untreated raisin separating the two treated ones with repellent was 10 ± 0. 3 mm. This greater distance was perhaps due to the factors of heat, CC>2 and probably skin odor, which were missing as attractant factors in the raisins.

TABLE 3 - Numbers oLA. aegypti settling on raisins separated by 1 cm, under the plain and repellent coated halves of a paper strip. Means of five replicates ± standard errors.

Paper strip half	Olive oil	D. M. P.	D. E. T.	Rutger 's 612	Indalone
Painted with
chemical	o r— H +1 00 j— H	5 ± 0. 5	3 ± 0. 7	3 ± 0. 1	2 ± 0. 8
Plain	19 ±4	18 ± 1. 9	12 ± 1. 3	13 ±1.4	10 ± 2. 2

Experiment 3

Raisins were kept 1 cm clear apart from each other and alternate raisins were painted with repellent. Other factors were the same as in the previous experiments.

The mean numbers of mosquitoes that landed on the treated and untreated raisins are given in table 4, columns 4 and 5. The numbers of mosquitoes feeding or settling on the untreated raisins were still very low and no significant differ ence was found in the number of mosquitoes feeding on untreated raisins in this experiment as compared to the number of mosquitoes feeding on untreated raisins in the previous experiment.

Experiment 4

Only 5 raisins were taken and were placed 1 cm clear apart and the portion of wire between them was painted with repellent. Since the raisins were not painted with repellent in this experiment their number was reduced to five so that the number of mosquitoes landing on them could be compared with the number of mosquitoes landing on the untreated raisins in previous experiments.

The results are given in table 4, column 6. The comparison of results in table 4 shows that significantly more mosquitoes settled on raisins in this experiment than in experiments where- treated and untreated raisins were placed close to each other. This is perhaps due to the small surface area of wire between the raisins as compared to the much greater area of the raisins in the previous experiments. This

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would result in a much slower production of repellent vapor.

TABLE 4 - Numbers of A. aegypti settling on raisins in the presence of repellents. Means of five replicates ± standard errors.

	Raisins close together, alternate raisins painted with repellent	Raisins 1 cm apart, alternate raisins painted with repellent	Raisins 1cm apart &: the wire in be- tween painted with repellent
Chemical		Untreated	Treated	Untreated	Treated	Untreated
		raisins	raisins	raisins	raisins	raisins
Olive oil		16 ±2	14 ± 4	10 ± 0. 7	11 ±1.4	10 ± 1
D.M.P.		1	0	2 ± 0. 5	0	2 ± 1
Rutger's		1	0	2 ± 0. 8	0	3 ± 0. 5
612
Indalone		0	0	0	0	3 ± 1
D. E. T.		0	0	2 ± 0. 5	0	5 ± 0. 5

Mating

In Aedes aegypti "The stimulus which induces the male to copulate is the sound produced by the female during flight. " . odor plays no

part in the sexual behavior of aegypti ..." (Roth, 1948, pp. 284, 282) . Roth also observed that in Aedes aegypti the male is the aggressor and is attracted by the female in flight and that the female is passive and does not show any mating behavior similar to that of the male, "...never in our observations was a male seen to initiate copulation with a resting female" (Roth, 1948, p. 276). Banks (1908, p. 246) on the contrary stated that specimens of aegypti confined in small jars "...have been seen to copulate while the female hangs from the gauze covering the vessel, the male always approaching her from the ventral surface. " Christophers (I960, p. 502) observed that copulation takes place quite commonly with the female at rest.

During the course of this work it was observed that a female Aedes aegypti is not entirely passive and that copulation does take place when a female is at rest. It was observed that when a flying male came close to a sitting female, the female would take flight and the male would grasp her for copulation. Many times females were seen taking flight spontaneously and males were seen getting hold of them in mid-air. The males were also observed coming to land sideways with a female,

14

Repellent Effects

then trying to take a ventral position and many a time they succeeded. At other times because of his efforts to gain a ventral position to the resting female the male roused the female to fly and copulation took place on the wing or the two could be seen falling to the floor copulating. But mostly copulation took place with a female in flight.

Roth (1948) also observed that the male would copulate repeatedly with the same or different females. After repeated matings, females become more and more reluctant to fly and would resist the attempts of the males to copulate. Richards (1927) suggested that repeated copulations exhaust the individuals. Shannon and Putnam (1934) in their laboratory study of A. aegypti observed that the average pupal period of females was 14 hours longer than that of males. Roth (1948, p. 308) observed that by the time the female begins to fly and becomes ‘attractive1 the male’s antennae have reached a state where the sound stimulus can be perceived and his genitalia have rotated sufficiently so that copulation can be successful (usually about 15 to 24 hours after emergence). In view of these observations it was neces sary in this work to separate the sexes before they started mating and to keep the observation time reasonably short. To forestall fatigue in the females due to repeated copulations, the males were separated from the females 14 hours after emergence.

Ten females 2-4 days old and 10 males 5-6 days old were used for each experiment. The females were chilled in a sucking tube for 1. 5 minutes at 15 F and then all their tarsi were painted with repellent with a fine brush while on a cold petri dish under a binocular microscope. Since Aedes aegypti mate venter to venter and the female does not clasp the male to her, her legs remaining out- stretched and serving as structures to which the male clings (Roth, 1948, pp. 27 0, 301), it was decided to paint the tarsi of the female mosquito with repellent. After the tarsi were painted the females were released in a one cubic foot cage and allowed to recover from chill. They recovered in 3-4 minutes. Ten minutes after the treatment 10 males were released in the cage. After application of the repellent on the tarsi of the female Aedes aegypti few flew spontaneously. Most females sat quietly on the walls of the cage. Males hardly ever succeededin persuading the female at rest to copulate. It was also observed, though no quantitative basis could be laid down for this, that the efforts of the male to copulate with the resting female, as well as with the female in flight, were less persistent and quite often they were seen releasing the female soon after coming in contact. The cage was therefore shaken every minute to make the females fly and the number of matings in a period of 30 minutes was recorded. Each experiment was performed with a new batch of mosquitoes.

The results are recorded in table 5. The standard error of the mean was used as a test of significance. The highly significant reduction in the number of matings in A. aegypti in association with repellents can be explained as a result of two factors: 1) a decrease in the flying

activity of the females and 2) less persistent efforts by males and premature release of the female.

Though the cage was shaken every minute in experiments with repellents as well as in the control it was observed that the females in

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15

the controls continued to fly for a much longer time after shaking than in experiments with repellents. With repellents, most of the time the females could be seen coming to rest on the wall immediately after shaking the cage, and many a time on shaking they would fly only from one wall of the cage to another. There was also a lack of spontaneous flight activity on the part of the females.

TABLE 5 - Numbers of matings in a 30 minute period in a population of 1 0 male and 1 0 female _Aedes aegypti with repellents applied to the tarsi of the females. Means of four replicates ± standard errors.

Control	Olive oil	D. M. P.	Indalone	Rutger 's 612	D. E. T.
65 ± 2	65 ± 1	33 ±2	30 ± 3	33 ± 3	33 ± 3

Oviposition

Wallis (1954) in his studies on the oviposition activity of mosquitoes, including A. aegypti , found that the female could detect an objectionable amount of salt even when the movements of the abdomen were restricted. Likewise surgical removal of the palpi, proboscis, and antennae from the head did not result in loss of sensitivity. Surgical removal or wax coating of various combinations of legs and leg articles resulted in the demonstration that sensitivity was localized in the tarsal articles of all the species of mosquitoes studied by him. His investi- gations also showed that the sensitivity was present in all the tarsal articles of Aedes aegypti. The thin walled chemor eceptor s of the tarsi enabled the mosquitoes to detect differences in saline concentrations as slight as 0. 02 M.

Browne (I960) studied the role of olfaction in the stimulation of oviposition in the blowfly Phormia regina Meigen. He found that the odor of a liquid medium containing powdered milk and yeast stimulated the blow- fly to oviposit. He also provided evidence for olfactory perception by the ovipositor of the blowfly.

In this study oviposition in Aedes aegypti was obs er ved by as s ociating potential oviposition sites with repellent vapors as well as by applying repellents on the tarsal chemor eceptor s.

Experiment 1

Five, 7-8 day old blood fed females in a one cubic foot cage were taken for each experiment. The cage was provided with a rectangular platform, 7 " x 4 " made of a steel wir e frame (diameter of wir e 2 . 5 mm). The platform was covered with nylon net on one side and with two paper towel strips pasted on the other except in the center where a gap of 1

16

Repellent Effects

cm was left in between the strips, see figure 1.

The platform was placed in the cage, nylon net side upwards, the ends resting on two glass bottles filled with water. On the nylon net was spread a piece of cheese cloth, the two ends of which remained dipped in the water in the glass bottles. The cheesecloth was kept wet by capillary action by the water in the two bottles. One of the two paper strips was painted with repellent while the other was left untreated. Thus an oviposition platform for the mosquitoes was provided, one half of which had repellent vapor coming from underneath through the nylon screen, while the other half served as control. The nylon net under- neath the cheese cloth served as a support for it and did not allow it to come in contact with the repellent on the paper strip below but allowed the repellent vapors to pass through. Most of the eggs were found to be laid on the cheese cloth but some were laid on the paper strip. They were counted separately 72 hours after the blood meal, and the results are recorded in table 6. Four experiments were run with each repellent.

The behavior of Aedes aegypti during egg laying is described in detail by Wallis (1954). During the experiments it was observed that a female mosquito could sample the oviposition sites while on the wing and would land on the control half rather than on the repellent treated half of the oviposition platform. At other times when she landed on the repellent half she walked for a few seconds and then flew away and landed on the control side. This behavior demonstrates the function of olfactory receptors in the selection of an oviposition site when repellent vapors are associated with it. The complete absence of egg laying on the repellent coated as well as olive oil coated paper towels on the lower side of the platform seems to be the result of tarsal chemor eceptor s which select the suitability of the egg laying medium on contact. The significantly small numbers of eggs laid on chees e cloth on the repellent side as compared to the number of eggs laid on the control side show that Aedes aegypti rejects oviposition sites when these are as sociated with repellent vapor.

Experiment 2

Experiments were also conducted by painting the tarsi with repellent by the same technique as described in previous experiments and recording the number of eggs laid in 24 hours. Christophers (I960, p. 507) records that egg laying in Aedes aegypti usually begins on the after- noon of the third day from blood feeding, counting the day of feed as zero. Female mosquitoes 6-7 days old were fed on blood and left in a cage with raisins for three days. On the fourth day their tarsi were painted with repellent and the mosquitoes were placed singly in separate vials with water soaked cottonwool in the bottom and a nylon net cap on the top on which was placed a raisin. Eggs laid in a 24 hour period were then counted. Four replicates were run for each experiment. The mean numbers of eggs laid are recorded in table 7.

The difference in the number of eggs laid in the control and those laid by repellent treated mosquitoes is not significant, using standard error of the mean as a test of significance. This is perhaps

17

Oviposition platform

Oviposition p latform - frame of steel wire (2.5mmqauqe)

b.

Cheese cloth

Nylon net pasted on oviposition platform

Water bottle

Paper towel strips pasted on oviposition platform-frame

Figure 1 . Diagrams showing: (a) arrangement of the oviposition platform in the cage, and (b) a vertical section of the oviposition platform.

18

Repellent Effects

due to the fact that the mosquitoes had no opportunity to select a site for oviposition as. they were confined in small vials.

TABLE 6 - Numbers of eggs laid by A aegypti females in the presence of repellents. Means of four replicates ± standard errors.

Chemical	Eggs laid on On cheese cloth	control side On paper towel	Eggs laid on On cheese cloth	repellent side On paper towel
Rutger 's 612	128 ± 18. 6	52 ±4.2	0	0
D. E. T.	160 ±20.5	47 ±2.2	2 ± 1. 7	0
Indalone	156 ± 18.4	19 ± 1.5	0	0
D. M. P.	208 ± 13	22 ±1.7	5 ± 1. 1	0
Olive oil	87 ± 6	12 ±1.1	68 ±7.2	0

Experiment 3

Experiments were also conducted to determine whether anten- nectomized mosquitoes would discriminate between the control and the repellent sides of the oviposition site . Twenty female mosquitoes which had been fed on blood previously were operated upon for each experiment on a cold petri dish under a binocular microscope after first chilling them for 1.5 minutes at 14°F. Ten to 12 flagellar segments of the antennae were excised and the mosquitoes then released in the cage with the oviposition platform shown in figure 1.

Though sometimes mosquitoes could be seen sitting on the control side of the egg laying platform, no eggs were laid in any of the experiments over a week's timeexceptin the experiment with diethyl toluamide where there were 4 eggs on the control side. A high mortality (70-75%) was also observed in mosquitoes during this period. The almost complete absence of oviposition by antennectomized mosquitoes may be due to lack of orientation of mosquitoes to the water soaked cheese cloth on account of the great reduction in the number of hygr or eceptor s as a results of excision and consequently a great increase in the threshold of moisture perception. The high mortality rate can also be assigned to the same factor, i. e. , lack of orientation to the water soaked cheese cloth and hence dehydration. Mosquitoes were seldom seen sitting on the wet cheese cloth. Most of the time they were found sitting on the walls of the cage with very little flight activity. The very low activity in antennectomized mosquitoes confirms the findings of Bar-Zeev (I960) who found only 4 per cent of mosquitoes could be activated when anten- nectomized as compared to 60. 1 per cent when intact.

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TABLE 7 - Numbers of eggs laid by single A. aegypti females after painting the tarsi with repellents. Means of four replicates ± standard errors.

Control	Olive oil	D. M. P.	Rutger 's 612	D.E.T.	Indalone
39 ± 8. 8	42 ±4. 8	29 ± 5. 2	34 ± 8. 6	34 ± 5	33 ±4.4

Experiment 4

Experiments were also conducted to test oviposition after treating the terminalia of the females with repellent. The female aegypti mosquitoes were fed on blood when 7-8 days old, and their terminalia painted with repellent 72 hours after the blood feed by the same technique as described in the previous experiments, and then released in the cage.

All the mosquitoes became too crippled to move about or fly shortlyafter the painting of the tip of the abdomen and died in a few hour s .

Behaviour in Relation to Wind Direction and Speed

Kalmus and Hocking (I960, p. 21) conducted a series of experi- ments in which target areas were drawn out on the backs of subjects who wore shirts with the backs cut out. They recorded the distribution of bites in relation to a small repellent treated area. To demonstrate the effect of wind direction on the distribution of bites in relation to repellent, experiments were conducted in the laboratory on Aedes aegypti using the same technique.

Experiment 1

A circle of 3. 5 cm radius was drawn in hard clear nail varnish on the bare chest of a subject. Concentric to this another circle of 6. 5 cm radius was drawn. The outer circle was divided into two equal halves by drawing a diameter. A hair drier was used to produce the air current and a variable transformer was included in the circuit to permit adjustment of the speed of the wind. The wind speed was kept at 43 cm/ sec and its direction at right angles to the drawn diameter. The source of wind, i. e. , the nozzle of the blower was kept 23 cm away from the central circle which was coated with repellent. The blower was kept in such a position as to give a uniform flow of air over the marked area. The repellent used was diethyl toluamide. One hundred 7-8 day old female Aedes aegypti mosquitoes were taken in a one cubic foot cage of steel wire with nylon net around it for each experiment. The mosquitoes were fed on sugar solution only before the experiment. The cage was placed on the marked area and the portion of skin outside the marked area was covered with a polyethylene sheet. Mosquitoes soon started biting through the nylon net on the floor of the cage. An observer kept a record of the mosquitoes that settled and flew away, or settled and bit, in the upwind and downwind halves of the circle. The counts

20

Repellent Effects

were made for 5 minutes in each experiment. Controls were run with the same wind speed without repellent. The number of mosquitoes that settled or bit in the upwind and downwind halves of the circle are given in table 8.

The results show that in the control where repellent was not painted in the central circle, significantly more mosquitoes settled or bit on the downwind side of the circle than on the upwind side. This is in conformity with the obs ervations made by Kalmus and Hocking (I960, p. 4) with field mosquitoes. However, when repellent was painted in the central circle it was observed that the number of mosquitoes settling or biting on the downwind half of the outer circle was significantly lower than the number settling or biting on the upwind half. This was due to the presence of repellent vapor carried by the wind on the downwind half of the outer circle.

TABLE 8 - Numbers of A. aegypti lemales settling or biting in relation to wind direction and D. E. T. on the marked area of skin.

	Control*	D. E. T. **
Wind speed	Upwind	Downwind	Upwind Downwind
43 cm/ sec	15 ±2. 5	27 ± 1. 6	35 ± 2. 9 8 ± 1. 7

*■ Means of two counts ± standard error ** Means of three counts ± standard error

Experiment 2

In another set of experiments the effect of different wind speeds was determined on the settling and biting of mosquitoes in relation to repellent. Experiments were conducted in a similar fashion as described under the experiments with different wind directions, except that the portion of the body used was the thigh instead of the chest, which gave the advantage of the subject himself making notes of the number of mosquitoes landing or biting. A control was run with each wind speed and all the controls with different wind speeds were run first in order to avoid contamination of skin area with repellent vapors. After the controls were run, different batches of mosquitoes were then used in experiments with the same wind speeds in relation to repellent painted in the central circle. The repellent used was diethyl toluamide. The portion of skin outside the outer circle was covered with polyethylene sheet and the count of mosquitoes settling or biting in the upwind or downwind half of the circle was recorded for five minutes in each experi- ment.

The results are shown in table 9. In previous experiments with different wind directions the number of mosquitoes settling or biting in the upwind half of the circle in experiments with repellents was

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significantly higher than the number of mosquitoes in the downwind half of the circle. The results given in table 9 show that the mosquitoes continue to showthe strong tendency of settling more on the upwind side in relation to repellent with different wind speeds.

The maximum wind speed at which mosquitoes were able to settle on a bluff body was reported to be 95 cm/ sec and that of settling on the streamlined body to be 55 cm/ sec. Kalmus and Hocking (I960, p. 15). In this case the maximum speed of wind at which the mosquitoes settled on the skin was 265 cm/ sec which is very high as compared to the wind speed with the bluff or streamlined bodies. This is probably due to the attractant factors of the skin acting on the mosquitoes.

TABLE 9 - The number of A. aegypti females settling or biting in the up- wind or the downwind half of the circle marked on skin in relation to different wind speeds and diethyl toluamide.

Wind speed	Control Upwind Downwind	D. E. Upwind	T. Downwind
0 cm/ sec	27	29	23	19
43 cm/ sec	13	26	41	12
1 34 cm/ sec	16	31	9	4
190 cm/ sec	5	14	3	1
227 cm/ sec	4	8	5	0
265 cm/ sec	4	6	2	0
314 cm/ sec	0	0	0	0

Orientation to Gravity and Centrifugal Force

Experiment 1

To study the orientation of Aedes aegypti to gravity in relation to repellents, experiments were conducted in a plastic petri dish of 9 cm diameter. The lid of the petri dish was perforated with 2 mm diameter holes, about 9 holes per sq cm to allow the repellent vapors inside the dish to escape. The floor of the petri dish was lined with a filter paper which was divided into four quadrants designated top, left, bottom, and right.

Twenty female mosquitoes, 7-8 days old were taken, chilled for 1.5 minutes at 14°F and then released in the petri dish. On recovery

22

Repellent Effects

of mosquitoes from chill the petri dish was turned with a diameter vertical and given five complete turns on the horizontal axis through its center; thereafter the position and the number of mosquitoes was noted in each quadrant after a minute. The experiment was replicated five times without repellent as a control. A band of repellent 1 cm wide was then painted on the outer margin of the top quadrant. Mosquitoes were chilled and placed in the petri dish and allowed to recover. After the mosquitoes had completely recovered, the dish was given five complete rotations as in the control, keeping it vertical and rotating it about its horizontal axis. The experiment was repeated five times with each repellent.

In the control the mosquitoes could be seen walking upwards and most of them collected in the top quadrant. Significantly les s mosquitoes remained in other quadrants . Almost all the mosquitoes were seen facing upwards and the root mean square deviation of their body axes from the vertical axis of the petri dish was found to be zero.

With repellent significantly less mosquitoes entered the top quadrant. Most of them remained in the left, right, g.nd bottom quadrants . They were also seen walking at an angle to the repellent or turning away from it. Their angle of turning (i. e. , the angles which the longitudinal axes of the bodies formed with the vertical axis of the petri dish) was noted by marking their position in each quadrant on a separate sheet of paper and then measuring the angle and direction of inclination to the vertical.

Table 10 shows the distribution of mosquitoes in the various quadrants of the petri dish in the presence of repellents, and table 11 shows the root mean square of the angle of inclination of the body axes of mosquitoes to the vertical in the presence of repellents in the petri dish.

Results with olive oil werenot found to be significantly different from those of the plain control.

The effect of the presence of repellent on the head upwards orientation of the mosquitoes in relation to gravity was highly significant.

Experiment 2

The effect on geotaxis of painting repellent on the mesonotum and the antennae was also observed. Seven to 9 days old female mosquitoes were chilled for 1.5 minutes at 14°F and their mesonotaor antennae were painted with repellent. They were then placed in a 9 cm petri dish having holes in the lid and lined with filter paper. After complete recovery of the mosquitoes from chill the dish was held with its central axis horizontal and rotated slowly about this, one rotation in 2 0 seconds, and the positions of the mosquitoes were noted. Normal female A. aegypti show a counter rotation to maintain a head upward under these circumstances (Kalmus and Hocking, I960, p. 8).

The mosquitoes with their mesonota painted oriented facing up- wards by counter rotation but when the antennae were painted with repellent, on placing the dish in a vertical position the mosquitoes could be seen sitting on the vertical surface head upwards cleaning their antennae with the tarsi of the forelegs. When the dish was rotated slowly

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while they were cleaning their antennae, they did not react until they faced downwards. Then they were found to lose their balance and were seen to place their forelegs on the vertical surface. Some of them turned around, faced upwards and started cleaning the antennae again, but typical counter rotation was absent.

TABLE 10 - Numbers ol A. aegypti females found in different quadrants in relation to repellents. Means of five replicates ± standard errors.

Chemical	Top	Quadrants Left	Bottom		R ight
Control	15	± 1	2 ± 0. 5	1	± 0.4	2	± 1
Olive oil	13	±1.6	3 ± 0. 8	2	± 0. 5	2	±0.7
D. M. P.	3	±0.4	4 ± 1	4	± 1	9	±1.3
D. E. T.	2	± 0. 1	6 ± 0.8	6	±1.4	6	± 0. 8
Indalone	4	± 1	5 ± 1.2	6	±1.4	5	±1.3
RutgerIs 612	2	± 0. 5	5 ± 0.4	8	±1.3	5	± 1

Experiment 3

According to Kalmus and Hocking (I960, p. 8), when mosquitoes were centrifuged in a 9 cm petri dish at 390 rpm and observed under stroboscopic illumination, they were found facing towards the center of the dish, and sometimes walking towards it.

In this study of the same behavior in relation to repellents a plastic petri dish of 9 cm diameter was lined with filter paper on which one radius was drawn in ink. Its lid was extensively perforated by small holes. Mosquitoes, bothmalesand females (50 to 60 adults) were released in this dish and centrifuged at 390 rpm on a turntable and observed under stroboscopic illumination. Mosquitoes were seen as reported by Kalmus and Hocking (I960) facing towards the center and walking towards it. Most of them collected near the center roughly 1 to 1 . 5 cm from it; fewer mosquitoes remained at the periphery. The centrifugal force at 1 cm from center was 1. 7 g and 1. 5 cm 2. 5 g. As the dish continued to rotate more mosquitoes could be seen moving towards the center. For experiments with repellents the mosquitoes were taken in batches of 15, in a sucking tube, chilled for 1. 5 minutes at 14°F and then their mesonota painted with repellent on a cold petri dish under a binocular microscope. All four repellents were tested. After treatment the mosquitoes were released in a cage and allowed to

24

Repellent Effects

recover. They were then introduced in the petri dish (50-60 of them) and made to rotate.

Under stroboscopic illumination it was observed that the mos- quitoes did not collect in greater numbers near the center of the dish and the movement towards the center was less noticeable. The dish gave an appearance of a scattered distribution of mosquitoes as compared to a circular distribution near the center in the control. Quite a few (10-15%) faced directions other than the center.

TABLE 11 - Root mean square of angles of inclination of the body axes of. A. aegypti to the vertical in the presence of repellents in a rotated petri dish in degrees. Means of five replicates ± standard errors.

	Control	D.M. P.	Repellents D.E.T. Rutger's 612 Indalone
Angle in degrees	0	43 ± 8.4	47 ± 13.3 50 ±5.4 47 ±13.3

In another experiment the mosquitoes themselves were not treated but a disc of 4 cm diameter (centrifugal force 3.5 g) was painted with repellent in the center of the dish. Mosquitoes (50-60) were introduced in the aish which was then rotated. It was observed that with an exception of one or two the mosquitoes remained outside the disc, sometimes facing towards it and sometimes turning away from it or walking around it. In yet another experiment when the diameter of the circle painted with repellent was increased to 6 cm (centrifugal force about 5 g) in 9 cm petri dish the same behavior was observed. Most of the mosquitoes remained outside the circle, although the non-treated peripheral belt around the repellent coated circle was only 1. 5 cm wide.

Yisual Responses

The optomotor and visual responses of mosquitoes have been studied by many workers. Kalmus (1958) reported that A. aegypti shows responses to the rotation of the plane of polarization of light. In a later study Kalmus and Hocking (I960, p. 19) observed swarming flight in A. aegypti close underneath a weak light sour ce placed on top of a darkened cage, but the same was not observed when a much stronger light was made to pass through a red filter. Mosquitoes were also observed by these workers to aggregate near the margins of black objects when these were placed on top of a weakly illuminated cage.

The visual response of mosquitoes was also studied by Kennedy (1939) and Rao (1947). Kennedy reported that suspended mosquitoes orientated accurately towards a vertical black stripe on a white back- ground. Presented with two stripes the mosquitoes faced one or the other stripe and not between the two. Rao ( 1 947 ) reported similar findings with Anopheles maculipennis atroparvus van Thiel, and Culex (Culex) molestus F orskal

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rendered flightless by the removal of the wings or by sticking them together .

To test the effect of repellents on the visual respons e of Aedes aegypti to black stripes, 20 female mosquitoes were taken in a glass bottle 12 cm tall and with a diameter of 3 cm. The inside of the bottle was lined with white nylon net to give the mosquitoes a good foothold. This bottle was placed inside a glass cylinder 14 cm high and with a diameter of 6 cm. The bottle and the cylinder were placed on a thick glass plate which was resting on a tripod stand. Under the glass was placed a 40 watt electric lamp which was covered all around with a cylinder of black paper so that light could go only upwards and light the bottle and the cylinder outside it uniformly from inside. In order that the inside of the cylinder be evenly illuminated, a filter paper was placed on the glass plate on which the outer cylinder and the inner bottle rested. The outer cylinder was divided into four quadrants and the alternate two quadrants were covered with black paper strips, each covering 90°. The remain- ing two quadrants were left uncovered, (figure 2).

As the outer cylinder was placed around the inner bottle contain- ing mosquitoes and kept there for a short time, the mosquitoes inside moved and came to rest on the wall of the bottle facing the black stripes. The outer cylinder was then rotated 90° so that all the mosquitoes now faced uncovered portions of the cylinder. The mosquitoes moved again in the direction of the black stripes and again came to rest opposite to them. This behavior could be observed again and again. However, when the antennae were painted with any of the four repellents they showed complete indifference to the black stripes and did not move towards them as in the control.

The experimental data on the effects of repellents on behaviour are summarized in table 1 1A.

TABLE 11A - Summary of data on the effect of repellents on responses to stimuli.

Table / Page	Response	Effect
3/12 & 4/13	Sugar feeding	Inhibition
5 / 15	Mating	Partial Inhibition
6/18 & 7/19	Oviposition-site treated -tarsi treated	Inhibition No Inhibition
8/20 & 9/21	To wind	Partial Inhibition ( D. E. T. only)
10/23 & 11/24	Gravity	Inhibition
/ 24	Optomotor	Inhibition

26

Outer cylinder

Black paper strips pasted on cylinder

Figure 2. Diagrams showing: (a) a vertical section of the apparatus used for testing the visual response of A.aegypfi females to black stripes in relation to repellents, and (b) a cross section of the outer cylinder.

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EXPERIMENTAL - LIPOID SOLVENTS

Amongst the advocates of chemical theories referred to in a previous section, many have suggested lipoid solubility as a basis of olfaction (Cohn, 1924; Dyson, 1938; Dethier & Chadwick, 1947; Dethier, 1948). Experiments were conducted to examine the effect of fat solvents applied on the antennal chemor eceptor s of Aedes aegypti females on their behavior towards a host.

Ten female Aedes aegypti eleven days old were taken for each experi- ment. The mosquitoes , which were fed on sugar solution only, were taken in a sucking tube and chilled for 1. 5 minutes at 15°F. They were then placed on a filter paper on top of a cold petri dish and their antennae were washed with lipid solvents applied with a fine camel hair brush. The mosquitoes were then transferred to a clean petri dish lined with filter paper in a one cubic foot cage and allowed to recover. Thirty minutes after the operation a hand was introduced into the cage and the number of landings of mosquitoes on it was recorded for a period of 15 minutes. Mosquitoes were shaken off gently on landing and were not allowed to feed on blood. The antennae of controls were rubbed with a clean dry brush.

The observations are recorded in table 12, and show that the number of landings decreased very significantly on washing the antennal chemor eceptors with the lipid solvents. But whether the decrease in landings is due to the loss of lipids from the chemoreceptor s , or due to the narcotic, anesthetic, or other effect of the solvents is uncertain.

TABLE 12 - Numbers of A edes aegypti females landing on a hand in a 15 minute period after treatment of the antennae with lipoid solvents. Means of three replicates ± standard errors.

Control Acetone Ether

174 ±8 52 ±12 43 ± 13

DISCUSSION

The action of repellent chemicals on mosquitoes has no specificity for blood feeding behavior. It has been shown that repellents in the vapor phase have the following effects on .Aedes aegypti . They inhibit feeding on both blood and sugars, reduce the mating rate, and cause rejection of oviposition sites. The repellents also affected orientation to gravity and centrifugal force and the visual response to black stripes.

Mosquitoes became quiescent and less active when repellents were applied on them. This slowing down of motor activity suggests the external stimuli normally acting on the mosquito are perhaps blocked or interfered with by the repellent. As there is no delay in the effect of repellents on the behavior of mosquitoes, that is, protection is obtained immediately these materials are applied, their action on the insect may be assumed to occur at the surface of the body. Repellents have not been shown to penetrate rapidly into the body where they could act on

28

Repellent Effects

the nerve synapses or the central nervous system, nor have they been shown to affect the muscular system directly. It thus seems unlikely that they act by blocking the nerve impulses or the motor response. The most probable action seems therefore to be the blocking of reception of stimuli at the receptor site.

Somewhat different behavior in relation to repellents of another kind has been described by Kennedy (1947). He studied the effects of contact with DDT on the activity and distribution of mosquitoes. He argued from his experiments that a variety of reactions may give rise to repulsion. Reactions may occur at a distance or only after contact with a repellent surface. The contact stimuli may be mechanical or chemical. The reactions may take the form of an increase of merely random activity or they may be directed away from the surface. They may be quick or slow to appear and weak or strong in expression. In contrast to my findings of reduced activity in his work an increase in activity was found.

The factor s that attract mosquitoes to the host have been reviewed above. The mode of action of insect repellents can be best understood when studied in relation to these factors.

The effects of repellents on the evolution of carbon dioxide and moisture from a human arm, and the correlation of this evolution with the natural attractiveness of human beings and protection time of repellents were studied by Gouck and Bowman ( 1959) at Orlando, Florida. In their experiments, repellents applied to the arms of three subjects reduced the CO2 emitted by 9 to 14 per cent but they concluded: ''Although these reductions are considerably greater than the differences between untreated arms (4%) they are not great enough to indicate that the mode of action of these repellents is based upon the retardation of carbon dioxide evolution". The repellents used were, dimethyl phthalate, diethyl toluamide and ethyl hexanediol. With regard to the moisture collected from untreated and repellent treated arms they concluded: "The quantities from the arms of all subjects varied from day today but in most individual tests the two arms agreed within about 5 per cent indicating that no real difference in the amount of moisture evolved was caused by application of repellents. " They believed that the protection time is governed by the rate of loss of repellent from the skin by absorption and evaporation. Peters and Kemper (1958) have shown that there are no considerable temperature differences between repellent treated and untreated parts of the skin.

In the light of these findings it can be said that repellents affect the reception of these stimuli rather than the stimuli themselves. This supports the hypothesis advanced that repellents affect many kinds of behavior of mosquitoes by interfering in the reception of many different kinds of stimuli.

Search for chemical factors other than carbon dioxide attracting mosquitoes to the host has claimed the attention of many workers. The findings of Shaerffenberg and Kupka (1951) and Bur ges s and Brown (1957) have indicated that attractive factors other than carbon dioxide are present in the vapor from mammalian blood and body exudations. A distillate obtained from mammalian blood by Shaerffenberg and Kupka

Khan

29

(1959) proved highly attractive to Culex pipiens L. Rudolfs (1922) found benzoic acid, dilute ammonia, phenylalanine, alanine, aspartic acid, cystine, and hemoglobin to be attractive to Aedes sollicitans Walker and Aedes cantator Coquillett, but Reuter (1936) found the last six materials unattractive to Anopheles maculipennis atroparvus . Brown and Carmichael (1961) reported that E-lysine and L-alanine wer e attractive to Aedes aegypti. The effect of repellents in association with these chemicals found to be attractive remains to be studied.

Travis and Smith (1951) evaluated dimethyl phthalate, indalone, and ethyl hexanediol against Aedes aegypti besides other mosquitoes, and found average repellent times (i. e. , times in minutes from application of the repellent to the first bite) as follows: ethyl hexanediol - 331

minutes, dimethyl phthalate - 247 minutes, and indalone - 111 minutes. Although the results of my experiments are not strictly comparable with those of Travis and Smith (1951) for I worked with a different culture of mosquitoes and at a different time and place, the mosquitoes fed on blood much sooner after treatment when repellents were applied on the mosquito receptor sites. For example, about33 per cent of mosquitoes fed on blood within 10 minutes after application of dimethyl phthalate on both antennae. When diethyl toluamide, indalone, and ethyl hexanediol were separately applied on both antennae, some of the first bites were recorded after 10 minutes. The reason for this behavior is perhaps the more rapid adaptation of the receptors'to the repellents because of the greater concentration gradient resulting from their application on the receptors themselves. In this way the threshold for reception of re- pellents increased greatly but that for other stimuli remained the same. The s equence of stimuli and responses leading to blood feeding therefor e remained unaffected. But this is, of course, incompatible with the hypothesis that repellents block all receptors.

The presence of separate chemor eceptor neurons mediating acceptance and rejection is assumed from the study of labellar chemo- receptor cells of Phormia regina . These cells have been the subject of co- ordinated behavioral, histological, and physiological study. A chemo- sensory hair of the labellum of this blowfly was described by Dethier (1955) as a hollow extension of the body cuticle possessing two distinct lumina. The chemosensory hair has been shown to be associated with three bipolar neurons, two of which send distal fibers to the terminal papilla by way of the thick- walled lumen of the hair. Dethier (1955) concluded that one of these neurons mediates acceptance while the other mediates rejection. On electrophysiological studies one of the two neurons was later designated the L fiber (for large spikes which re- sponded to salts and the other the S fiber (for small spikes) which re- sponded to sugars (Hodgson et al. 1955; Hodgson and Roeder, 1956). Wolbarsht and Dethier (1958) were able to detect the spikes of the third neuron which terminated in a process at the base of the hair. It was designated M for mechanor eceptor . Evans and Mellon (1962) have now detected spikes from a fourth neuron which responds to water.

In the course of electrophysiological studies of chemor eceptor hairs it has been shown that when mixed stimuli are applied there is an interaction between activity in the L and S fibers (Hodgson, 1956 ,

30

Repellent Effects

1957; Morita, 1959; Sturckow, 1959). Hodgson (1957) found that the presence of S impulses is accompaniedby a decrease in L impulses and conversely the S spikes decrease when the L fiber is stimulated. My experiments show that the repellents block the reception of attract ant and other stimuli. This assertion needs to be confirmed by electro- physiological methods.

Mosquitoes with antennae painted with diethyl toluamide landed, walked around, and even probed on an arm also treated with the same repellent but did not feed on blood. This may be explained in one of two ways. It may be that the piercing of the skin by the mosquito is induced by some chemical factor on the skin which was neutralized by the application of the repellent or, it may be due to the effect of repellent on the action of thermoreceptor s or contact chemor ecptor s which in- duce feeding on blood. The latter explanation would be more in con- formity with the findings that repellents interfere with the reception of all kinds of stimuli affecting the total behavior of mosquitoes.

The study on blood feeding when repellents were applied on parts of the mosquito revealed that of the four repellents dimethyl phthalat e has the greatest effect on blood feeding behavior when it is painted on the tar sal receptor s and the smallest effect when it is painted on the receptors of the antennae. As is known, the olfactory receptors are located on the antennae and the contact chemor eceptors mostly on the tarsi of the mosquito. Dimethyl phthalate, which has the highest boiling point and hence the lowest vapor pressure, may, for this reason, have more effect than the other repellents through the tar sal chemor eceptor s in the liquid phase but less than these through the olfactory receptor s of the antennae where it has to act in the vapour phase which is at a lower concentration.

That repellents also acted as irritants was evident from the intense wriggling activity of the mosquito when repellents were applied on the proboscis and from the vigorous cleaning of repellent from the antennae with the tarsi of the fore legs. This evident awareness of the presence of an irritant chemical indicates the existence of receptors sensitive to it, perhaps those of the common chemical sense. It may well be that these are the only receptors not blocked by repellents.

In the vapor phase repellents were found to inhibit landing of mosquitoes. This was observedin experiments on blood feeding, sugar feeding, oviposition, and air flow. In the liquid phase, however, the repellents showed more irritant and some toxic effects, and the mos- quitoes showed considerable decrease in locomotor activity, in part on account of preoccupation with attempts at cleaning off the repellents.

Repellents have been defined as compounds which elicit an avoiding reaction (Dethier, 1956b). While the four materials studied may all do this, this is by no means their only effect and may not, in- deed, be the most important one.

Khan

31

ACKNOWLEDGEMENTS

I am most thankful to Professor B. Hocking for his keen inter est, constructive criticism and many valuable suggestions duringthe progress of this work. I also gratefully acknowledge the support of the Defence Research Board, Department of National Defence, Government of Canada, who financed this study.

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Repellent Effects

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36

Book Review

LINDROTH, C. H. 1963. The gr ound-beeltes of Canada and Alaska. Part 3. Opuscula Entomologica, Supplementum XXIV, pp. 201 - 408, Figs. 102-207. Zoological Institute, University of Lund, Lund, Sweden. Price - 35 Swedish crowns.

This portion of this work, the second to be published, includes the last part of the taxonomic treatment of the genus Trechus , and a rev- ision of the bembidiine genera Asaphidion Gozis (three species ), Bembidion Latreille, and the monotypic genus, Phrypeus Casey. The treatment of Bembidion occupies 200 of the 207 pages. This volume is based on an examination of the relevant material stored in the major European and North American museums and private collections, and on the extensive collections of Lindroth.

As in part 2, Lindroth provides for each species a succinct synonymy, a synoptic description, and data on type locality , ecology, and geographical distribution.

The text is straight - forward, simple English. The resulting clarity of expression illustrates very well the author's thorough knowl- edge of his subject.

The illustrations are excellent, and those of the entire insects are among the best ever executed of carabid beetles. For many of the species, the internal sac of the male genitalia, with its complex folds and peculiarly shaped sclerites is illustrated, in the infolded position. Also provided are simple, clear - cut line drawings of various other structures. All drawings were made by the author himself.

The treatment of the genus Bembidion is the dominant feature of this volume. The 193 species, 31 of which occur in the United States only (excluding Alaska), are arrayed in 48 groups. An additional six extra - limital species are included in the key to species, but are not treated elsewhere in the text. For each group, a brief diagnosis is given, as well as the subgeneric name that would apply if the author chose to use the category subgenus. Twenty-five new taxa are described, of which four are ranked as subspecies. Of the new species, the type localities of six are in the United States (excluding Alaska). Although the work deals primarily with the Canadian and Alaskan fauna, Lind- roth treated all of the known North American species for a number of the species groups.

Bembidion has long been regarded as the most difficult and complex genus of carabids in North America, and the justification for this opinion is perhaps best illustrated by the large number of synonyms listed - 165-of which 159 were proposed by one author , Colonel Thomas Lincoln Casey. (By way of contrast, 21 Casey species are recognizedas valid, and his names are also used fo-r another two species, as a result of the fir st-used names being junior homonyms). The synonymy is based upon study of the type specimens by Lindroth, and the facts should settle any doubt about the value and quality of Casey's work in the Carabidae. Hayward's revision of 1897 (Trans. Amer. ent. Soc. , vol. 24) was also grossly inadequate. Lindroth's extensive knowledge of the European

37

species of Bembidion , plus his thorough familiarity with Netolitzky's fine study are factors which contributed in an important way to the suc- cess of the study of the North American species . Thanks to this revision, it is now a relatively simple task to determine any specimen from Can- ada or Alaska.

The two keys for identification (one to species groups , and one to the species) are easy to use. This statement is based on personal experience gained by identifying several thousand specimens, represent- ing a substantial portion of the species. Each couplet in the keys consists of a pair of clear - cut alternatives , and there are no complicated "either- or” statements. One of the features facilitating use of the long key to species (225 couplets ) is that the numbers of those couplets which set off a large number of species are in bold face. In spite of these good features I have three criticisms to make regarding the keys: a. no attempt was

made to relate directly the species-group key to the species key; b. names of author s of species were not given in the key; c. page references to the text were not given for the Canadian and Alaskan species . However , these are minor points, and the last one is largely taken care of by the number which is assigned to each species in both key and text.

In a key of this length, it is almost impossible to avoid errors, and it is with regret that the following omissions of species are noted: 64. nigrum Say; the species of the incrematum group- 103 incrematum LeConte, 104. immaturum Lindroth, and 105. gracilitorme Hayward; and humboldtiense Blaisdell, p. 305.

The fact that only a few subspecies were described or recognized may suggest that the author is unaware of current taxonomic theory. Such, however, is not the case. Lindroth notes carefully geographical variation where he finds it, but he describes as subspecies only those populations which are clearly geographically isolated from their closest relatives, and which differ markedly from them. He avoids naming populations which are segments of dines, and thus avoids proposing a lot of trinominals which will subsequently have to be synonymized.

A search through the work for indications of modern techniques of analysis will prove fruitless. One does not find complex graphs, charts, or long tables, and only very few simple statistical parameters are indicated. However, the study does not suffer from this seeming lack. This seems to me to show that a major attribute of a good taxon- omist is the ability to interpret correctly carefully chosen, accurate observations. This is not to say that the study of the genus cannot be pursued profitably with more sophisticated techniques, but rather that I doubt that such techniques would have provided, at the present level of understanding, much more than Lindroth was able to state using the methods of analysis that were in use in the time of Linnaeus. This illustrates that the difference is unimportant between 'modern' as opposed to 'old fashioned' taxonomy; the distinction should rather be made between 'good' and 'poor' taxonomy.

R egarding clas sification of Bembidion , I think the author is mistaken inusing only a single infra-generic category, namely 'group'. In a genus of this size, several infra-generic categories are required to point out the similarities and differences among the species: subgenus, species

38

group and sub-group, at least, However, Lindroth states that such a classification should be proposed on the basis of a study of the world fauna, and perhaps he is right.

The work has, so to speak, opened the door to the study of North American Bembidion . It provides a basic classification, which can be easily modified, as required. It shows clearly how diver s e the genus is. The task of completing the revision of the North American species will be a pleasure. Because of the marked ecological specialization of many of the species, the genus should provide valuable material for the study of the origins of adaptations. Also, the numerous species and their wide distribution in North America, should provide fertile ground for the development of zoogeographic studies. And, returning to description of structures, one should remember that the immature forms are vir- tually unknown. Lindroth has provided an excellent platform from which to launch further studies, and it is to be hoped that such studies will be made in the near future.

Carl Lindroth brought to this work a feeling for these fascinating little creatures which is best described as deep affection. And this, combined with unrivalled knowledge, superb talent, and hard work on the part of the author , has provided us with the finest taxonomic treatment of a group of carabid beetles ever produced.

George E. Ball

Quaestiones

entomologicae

A periodical record of entomological investigations, published at the Department of Entomology, Uni- versify of Alberta, Edmonton, Canada.

VOLUME I

NUMBER 2

APRIL 1965

39

QUAESTIONES ENTOMOLOGICAE

A periodical record of entomological investigations, published at the Department of Entomology, University of Alberta, Edmonton, Alberta.

Volume 1 Number 2 6 April 1965

CONTENTS

Editorial . 39

Pucat - The functional morphology of the mouthparts

of some mosquito larvae 41

Editorial - Beastly teachers

Teacher s , they say, area necessary evil; beastly people, teacher s ; pedantic, dogmatic , intolerant. If this is the nature of the beast, should we not take Wordsworth's advice and 'let nature be our teacher'? There could be no better field than entomology in which to put this into practice; at least we should run no risk of a shortage of teachers.

It is difficult to arrive at a reasonable estimate of the world pop- ulation of entomologists, because they are difficult people to define and still more difficult to regiment (praises be]). If one supposed that for every one attending an International Congress, ten stay at home - or more likely go out collecting - there must be around 20, 000. If Canada has as many per head of population as any country, as has been claimed, the figure may be 50, 000. Let us average these two figures; if we have 35, 000 entomologists, this would allow 22 described species of insect per entomologist, or if we accept C. B. Williams' estimate of the world population of insects at 10^, about 3 X 10^ insects per entomologist; a rather unusual staff/ student ratio.

Insects are certainly pedantic, dogmatic, and intolerant, and should therefore make good teachers. And as teachers of entomology they must surely be immune to the fashionable accusation directed at school teacher s -that they are good teachers but have nothing to teach, if not to the reciprocal retort often aimed at university teachers. Perhaps this is the proper role of human teachers of entomology - to help the insect teach the student, or to help the student to learn from the insect. Cer- tainly if one had to choose between insects , books, and entomologists , from which to learn, the choice would be in the order given. Perhaps more than any other science, biology in general and entomology in particular mustbe taught from the organisms they are concerned with, in the field and in the laboratory. Many of us get into the bad habit of reaching for a text when in doubt about some point of insect structure, when we could just as

40

easily reach for an insect - a much less fallible adviser. The habitual reference of questions back to the insect might even help us in our dif- ficulties in keeping up with the literature; it would certainly give us a surer foundation of knowledge from which to judge whether, in any part- icular paper, we need to read on. In addition to the rather negative qualities we started out with, insects are ubiquitous, lively, versatile, unobtrusive, fertile, and unequivocal. There i s little more one could ask of a teacher.

One of the interesting advantages of an insect teacher of entom- ology as compared with a human teacher , is that he can fulfil many of his functions even after death, especially if well preserved. Indeed it is in large part the readiness with which they may be acquired in the first place and preserved in the last place, that makes insects so much more valuable than many other groups of organisms in the teaching of other bran- ches of biology. Their only limitation lies in their inability to teach the structural detail of other groups - unfashionable stuff these days anyhow.

There is a tradition of great teachers of entomology extending back to the early years of the science itself. Surely a place in this roster has been earned at least by two species of cockroach, by a fruit fly, and by mealworms and flour beetles.

Brian Hocking

THE FUNCTIONAL MORPHOLOGY OF THE MOUTHPARTS OF SOME MOSQUITO LARVAE

A.M.PUCAT „

Division of Natural Sciences Quaestiones entomolo gicae

University of Saskatchewan, Regina oo 1 O

Homologies of the parts of the maxilla and the labium of mosquito larvae were studied. The name cardobasistipes is proposed for the triangular sclerite latero-posterior of the maxilla, previously known as the cardo or the palpifer. The numbers of serrations on the prementum and submentum were found to be of taxonomic value. The sequence of mouthpart movements of filter feeding and browsing species, and the progress of food particles from the feeding current into the mouth were observed. Differences in stiffness were found among the setae in different posi- tions on the mouthparts. These differences were confirmed by staining the cuticle with Mallory’s triple stain and are correlated with the functions of the setae during feeding. Flexible serrations at the tips of the labral brush hairs are used for raking food particles in most of the browsing species of Aedes and Culiseta studied. When in pond water neither the browsing nor the filter feeding larvae select the type of food they ingest. Feeding behaviour of the predatory larvae of Chaoborus americanus (J ohannsen) and Mochlonyx velutinus (Ruthe) was observed.

INTRODUCTION

The mouthparts of a mosquito larva occupy a large portion of its head; their structure is degenerate. In this work emphasis is placed on the homologies of the parts of the maxilla and the labium, on the structure and function of the labral brushes and on the type and size of food part- icles ingested by the larvae.

The problems of homologies of the mouthparts did not occupy the early biologists who lacked adequate equipment for detailed study of minute structures. Hooke (1665) drew a mosquito larva, but he did not interpret all the parts of its anatomy accurately; for example, he labelled the external opening of the respiratory siphon as the anus. He further said about the "Water - Ins ect or Gnat": --"It is suppos'd by

some, to deduce its first origin from the putrifaction of Rain Water. . ." He wrote that the larvae can move gently through the water by moving their mouthparts, and "eat" their way up through the water.

Reaumur (1738) described and illustrated the external features of a mosquito larva which seems to be a Culex species ( pipiens according to Shannon, 1931). He gave an accurate description of the function of the labral brushes and described browsing and filter feeding activities of larvae.

The best known studies on mosquito larvae in the 19th century

42

Mouthparts of Mosquito Larvae

are those of Meinert (1886) and Raschke (1887) who discussed larval morphology, function of mouthparts, and some of the habits of larvae and adults.

The names used by authors for the mouthparts of mosquito larvae are summarized in table 1. The following author s also referred to some mouthparts by specific names: Miall (1895), Johannsen (1903), Mitchell (1906), Puri (1925), Montchadsky (1945), and Cook ( 1 956) . A more complete list of literature on this subject is included in my thesis (Pucat 1962). It is evident that there is disagreement on the homology and nomenclature of certain mouthparts. There is less disagreement on the function of these parts, but this has not been studied exhaustively.

Classification of Feeding Habits

The structure of mouthparts, the method of feeding, and the habitat of the larvae are inter - related. On the basis of these factors culicine larvae have been classified into filter feeders, browsers, and predators (Surtees 1959).

It has been found convenient to follow this classification since it is based on morphological and functional characteristics. The crit- eria may be summarized as follows;

Filter Feeders - are larvae which strain out food particles from the water, such particles being sufficiently small to pass directly into the digestive tract without undergoing any further breakdown. Their salient morphological characters are: long, fine, unserrated labral brushes,

large maxillae bearing many fine setae, small weakly chitinized man- dibles, a weakly chitinized submentum possessing a large number of very small teeth and, associated with these features, large sub-apical tufts of setae on the antennae (Surtees 1959). These structural features were recognized by Wesenberg-Lund (1920) in several Danish species of mosquitoes. Nuttall and Shipley (1901) described in detail the function of the labral brushes of a filter feeder, an unnamed Anopheles species.

Feeding action similar to that observed by Nuttall and Shipley was also observed by Bekker (1938a, b) in Anopheles maculipennis Meigen, and by R enn (1941) in Anopheles quadrimaculatus Say and Anopheles crucians Wiedemann. Renn referred to the characteristic anopheline feeding method in which the floating particles are drawn straight towards the mouth as "interfacial" feeding. However, sometimes anopheline larvae employ a feeding method common to the larvae of other genera of mos- quitoes in vhich the particles move in converging curved lines, and this Renn calls "eddy" feeding.

Browsers - abrade solid material, the particles of which require further manipulation by the mouthparts before entering the digestive tract (Surtees 1959). Mouthparts of this type have been describedby Mitchell ( 1906), Howard, Dyar, andKnab (1912), We senberg- Lund (1920), Surtees (1959), Snodgrass (1959), Christophers (I960), and Clements (1963). All authors agree that browsing larvae are usually bottom feeders.

The labral brushes as well as the maxillary andmandibular bris- tles are shorter and stiffer than in the filter feeders. As Mitchell (1906)

Pucat

43

pointed out, in brushing over debris at the bottom of a pool very long, slender hair s would be a disadvantage. Mandibles are used to manipulate any large particles that come into the feeding stream, and the submentum is used as a secondary grasping organ. The swimming position is usually at an angle of about 45 ° to the substratum. Morphological gradations occur between typical filter feeders and browsers ( Wesenber g- Lund 1920, Surtees 1959).

Predators - have the labral brushes strongly chitinized. The role of the maxillae has been suppressed and the mandibles are the principal mouthparts. These are very large with strongly chitinized claws and take upmostof the oral region of the head capsule. As sociated with the strong claws are large, stiff spines which also aid in grasping the prey. This is true of the larvae of Chaoborus and Mochlonyx (Schremmer 1950, Peterson 1951, Cook 1956, and others). The submentum in all predatory species is well developed, the teeth being large and generally pointed. The increase in the strength of the submentum is associated with a reduction in the number of teeth and mouth brushes. Predatory larvae have large prehensile antennae which aid in grasping prey.

Evolution

Montchadsky (1937) has considered the environmental adaptation of larval and adult structures and behavioral characteristics important in classification. The type of feeding is a factor correlating the processes of evolution of larval and adult mosquitoes.

The Anophelinae and Culicinae have mostly plant-feeding larvae and blood - sucking adults (Montchadsky 1937, Hennig 1950). However, the Toxorhynchitinae and the culicine subgenus Lutzia have reversed their type of feeding; the larvae lead a predatory life, but have structures which indicate a previous adaptation to a vegetarian type of feeding. The adults of these mosquitoes either feed on plant juices (but carry traces of previous ability to suck blood), or appear to be optional blood feeders (Montchadsky 1937). In the Chaoboridae the adults are plant feeding while the larvae are predatory. Two lines of adaptation to predation are known: the surface film feeders such as Eucorethra , and the pelagic

feeders such as Chaoborus .

In the initial stages of evolution of the mosquitoes either there was a change in the type of feeding of the adults (transition to blood feeding in the subfamily Culicinae), or of the larvae (the transition to predation in the Chaoboridae). According to Montchadsky (1937) these changes were provokedby certain changes in the nutritional requirements for the ripening of the sexual organs. If adequate food containing high quality protein is eaten by the predatory larvae, it is not then required to be eaten by the adults which may be vegetarian. On the other hand, non - predatory mosquito larvae do not obtain adequate high quality protein, so that the adults of these species must have it from the blood of vertebrates.

TABLE 1 - Summary of names which have been used for some mouthparts of mosquito larvae.

46

Mouthparts of Mosquito Larvae

MORPHOLOGY OF THE HEAD AND MOUTHPARTS OF MOSQUITO

LARVAE

The mouthparts of mosquito larvae were compared with the mouth- parts of larvae of other Nematocera, Mecoptera, and other panorpoid groups, or with published descriptions of them.

Procedures

Two species of mosquito, Aedes aegypti ( L. ) and Culiseta inornata (Williston) were reared in the laboratory, so that fresh specimens of these species were almost always available. Rearing methods of Trembley (1955) and McLintock (1952) were followed. Specimens from the field were also observed alive and dissected in the laboratory. Since larvae were available in abundance, dissected heads were mostly studied. The dissections were done in glycerine. Hoyer's mounting medium and neutral Canada Balsam were used for mounting the mouthparts. Eosin- water solution was used for staining dissected muscles , and modified (Peterson I960 ) Mallory's triple stain for larval head cuticle. The mouthparts were boiled for 15 minutes in an 8% aqueous solution of KOH before staining.

Manton ( 1958) commented on the staining reaction of cuticle with Mallory's. She concluded that sclerotized non- staining exocuticle is unstr etchable when thick, that orange and red- staining cuticle are progressively less fully slcerotized, less rigid, and more elastic than the non- staining cuticle, and that blue- staining cuticle is fully flexible, more stretchable, but less elastic.

The structure of the heads of the larvae of Aedes fitchii (Felt and Young) and Culiseta inornata was studied in detail, and other species (table 2) were compared with them. Larvae of a Chironomus species, and of Mochlonyx velutinus (Ruthe) and Chaoborus americanus (.Johannsen) were also examined.

The Head Capsule

The largest sclerite in the head capsule of a mosquito larva is the fr ontoclypeus , which extends over most of the head surface dor sally. The genae are lateral, the postgenae postero-lateral; they extend vent- rally to complete the head capsule (figs 1,2). The median ventral part of the united postgenae, posterior to the mouth, has been given various names. I consider it as the subgena. It is bounded by two lines of cuticular thickening ridges which are known variously as the submental- postgenal sutures (Shalaby 1956 and 1957a, b,c,d) hypostomal sutures (Menees 1958a, Christophers I960), and thickening ridges (Snodgrass 1959). I agree with Snodgrass' interpretation of the homologies of the ventral head sclerites. In homologizing these sclerites of the mosquito larva Snodgras s digresses to discuss the ventral head sclerites of other insects, especially insects in which a trend toward a ventral elongation of the postgenae is evident. As examples he cites certain beetles in which the entire labium with a gular addition to the submentum is enclosed between the postgenae. He states, however, that this condition is not

47

0. 5 mm

posterior

tormal

apodeme

labral brush flexors

hypopharyngeal bar

salivary duct

maxillary

pr emental

Fig. 1. The head of Aedes fitchii (F. & Y. ) larva, (a) dorsal view showing muscle origins and extended labral brushes, (b) ventral view with brushes retracted and mouthparts removed from right hand side. mx. maxillae, md. mandible, sm. sub- mentum, t.m. tessellated membrane, aul. aulaeum, p. t. posterior tentorial pit. Muscle attachments stippled.

48

a

0. 5 mm

Fig. 2. (a) Lateral view of the left side of the head of Aedes fitehii (F. & Y. ) larva,

(b) Sagittal section through the mouthparts of Aedes fitehii larva, md. mandible, mx. maxillae, pm. prementum, sm. submentum, aul. aulaeum, distist. dististipes. Muscle attachments stippled.

Pucat

49

r epresented in mosquito larvae . Mor e commonly , the postgenae come to- gether medially and displace the labium. A final stage in the displacement of the labium is seen in the larvae of Chironomidae where the labium has become greatly reduc ed and is hidden from below by a median hypostomal lobe of the united postgenae.

A similar proces s of closure and elongation of the postgenae and reduction of the labium occurs in nematocerous larvae as discussed by Anthon (1943), Hennig (1948, 1950, 1952), and Snodgrass (1959). In the larvae of the primitive rhyphid Olbiogaster the small postgenal lobes are posterior to the submentum of the labium (Anthon 1943). In tipulid larvae, described by Vimmer (1906) and other authors, as well as in other .iematocer ous larvae the genae are completely united ventrally and the labium is dorsal to the subgenal lobe. In the mosquito larva, to distinguish the central area between the thickening ridges of the genae Snodgrass (1959) named it the subgena, and the areas laterad of the ridges the postgenae. I use this nomenclature.

Cook ( 1944a, b, 1949), following Ferris's (1947) and Henry's ( 1 947) theories of the segmentation of the arthropod head, considered the postgenae and the subgena as parts of the maxillary segment. Shalaby (1957) considered the apical part of the subgena as the mentum and the remainder as the submentum. As evidence for this idea Shalaby referred to Wheeler's (1893) embr yological work in which the latter observed that the rudiments of the second pair of maxillae on the sides of the embryonic body give rise to the labium in the embryos of the locust Xiphidium ensiferum Scudder, in Gryllus luctuosus Serville, and in Stagmomantis Carolina ( Johanns en). Shalaby believed that the median suture present on the ventral sclerite of the head of Culex molestus Forsk. larva is due to incomplete fusion of the embryonic rudiments of the second maxillae. That the embryonic second maxillae give rise to the labium has been shown by Butt (1957) in Oncopeltus fasciatus (Dallas), and by other authors in other insects. Christophers (I960) also believes that the subgena is the labial area; he homologizes the subgenal and postgenal areas posterior to the maxillae with the fused bases of the maxillae (cardo and stipes). He thus believes that in the larval as in the adult stages of mosquitoes the bases of the maxillae extend to the occipital foramen, forming the hypostomal area. However, the sclerite which Christophers considers as the base of the maxilla serves as the origin of pharyngeal, man- dibular, and maxillary muscles which in most other insects originate on the tentorium or on the cranial wall (Snodgrass 1935). In the adult Aedes vexans (Meigen) the maxillary muscles originate on the tentorium (Peterson Hoyt 1952). On the other hand, none of the postgenal muscles of the mosquito larva originates on the tentorium. If the larval post- genaand subgena are to be considered as the fused maxillary cardo and stipes, then the origins of the various muscles upon them are difficult to explain. Menees (1958a), studying the embryonic development of A. quadrimac ulatus , observed that the median suture on the ventral head sclerite in this species is the result of incomplete fusion of the postgenae.

Most sutures which are characteristic of the primitive insect head are absent from the heads of mosquito larvae. Two cleavage lines extend anteriorly from a short posterior occipital stem (fig 1). These

50

Mouthparts of Mosquito Larvae

cleavage lines may be homologous with the frontal sutures and the epi- cranial suture of other insects. However, Snodgrass (1947, 1958) and DuPorte (1953) state that the frontal arms of this suture follow diverse paths in different insects, and therefore do not define any specific part of the head. For this reason, in this workhead sclerites and mouthparts have been named in reference to muscle origins.

Approximately in the center of the frontoclypeus arise the labral and epipharyngeal muscles (fig. 1) which usually originate on the clypeus, and posterior to these are the origins of the pharyngeal muscles which generally occur on the frons. In the head of Aedes fitchii (Felt and Young) larva and in all the other mosquito species examined, there is no demarcation between the areas where the different muscles originate. According to DuPorte (1962) in some insects the boundary between the clypeus and frons, in the absence of an epistomal suture, is fixed by the position of the anterior tentorial pits. In the heads of mosquito larvae, however, the epipharyngeal muscle (usually on the clypeus) orig- inates much posterior to the anterior tentorial arms.

The tentorium in the mosquito larva is represented by anterior and posterior arms. The anterior arms originate on the head capsule medial to the antennae, in the same area where the hypopharyngeal bars arise (fig. 1). The long, slender anterior tentorial arms connect to the short posterior arms on the postero - ventral part of the head. There is no tentorial bridge.

On each side of the head a hypopharyngeal bar connects the hypopharynx to the side of the cranium (fig. 1).

The Labrum

The labrum of the larva of Aedes fitchii consists of a narrow transverse sclerite dorsally (fig. 1). Ventrally it is composed of a membranous area to which three brushes are attached, one median and two lateral and movable. The median brush is connected to each lateral labral brush and to the distal part of the dorsal labral sclerite by a membrane which has ben variously named. In the larvae of Lutzia halifaxi Theobald, Cook (1944b) referred to it as a "pennicular area., beset with small oval pits arranged in definite rows. " Because of its appear- ance Christophers (I960) called it the tessellated membrane, and this is the name adopted her e (fig. 5). However, this name does not describe the membrane accurately in all the larvae that I examined. This is discussed further below.

In both A. aegypti , (Shalaby 1957a) and A edes fitchii , two types of hair s ar e found on the median brush; long thin br anched hair s posteriorly , and short stout hairs with serrated distal ends anteriorly. Both types are shorter on the sides of the brush than medially.

The lateral labral brushes are composed of three types of hairs which differ in length, thickness, curvature, and location. The hairs of the first type are simple, relatively short, thin, soft, without definite curvature, and are located postero - laterally, dorsally, and ventro- medially overhanging the pharynx (figs. 1,3). These hairs, which are attached to the tessellated membrane, do not take part in creating a feeding current. Hairs of the second type are long, simple, thin ,

Pucat

51

slightly curved at their bases and at their distal ends, and are located in the lateral posterior two thirds of the brush (fig. 3). Anterior to them are hairs of type three. Types two and three take an active part in creating currents. The apices of type three hairs are provided with serrations (17-20 per hair). The serrations on the lateral type three hairs are smaller and slightly closer to each other than those on the more medial hairs.

Three types of hairs were found in all the browsing species of Aedes and Culiseta except in Aedes cinereus Meigen and A. canadensis ( Theo). which have only short, simple hairs on their lateral brushes. When the labral brushes are stained with Mallory's the bases of all the hairs stain red. Next above the bases a narrow layer of blue appears across the hairs and above this layer hairs of type one and two stain red to their tips. Hairs of type three stain partly red above the blue portion but they stain blue apically, in their serrated regions. A large proportion of the most median type three hairs stains completely blue above the red bases. In A. fitchii and the other Aedes larvae, as well as in the browsing Culiseta larvae that were examined, the apices of hairs of types one and two are tapered. Also tapered are the apices of all the hairs ofthelabral brushes of the filter feeders, Culiseta mors itans (Theo.) and

Culex territans Walker. In the brushes of the filter feeding larvae all the hairs are simple. They all have red- staining bas es , blue- staining portions above the bases, and red- staining middle and apical portions. In the filter feeding larvae a large group of hairs, originating medially on each lateral labral brush, overhangs ventrally, partly covering the epipharynx, A smaller number of simple hairs extends in this position in the browsing larvae (fig, l),In all the larvae that were examined these hairs are red - staining. In the larvae of Chaoborus americanus the labral brushes consist of a fewhard, short, brown bristle s on the small sclerite. In the larva, of a Chironomus specie s examined a few labral bristles ar e red- staining and the remainder are blue- staining. Thus the staining reaction of the labral brushes of the filter feeding and browsing larvae indicates that their hair bases are elastic and the portions above the bases are flexible. Flexibility of these hairs was seen when larvae were observed feeding and also v/hen the hairs were deflected with a needle.

In the mosquito larvae examined all the hair s of the lateral brushes except type one are attached to sclerotized rods which extend transversely across the basal area of the brush (figs. 3 and 4). Salem (1931) seems to be referring to these rods in Aedes fasciata (Fab.) ( A. aegypti L. ) when he states that the chitin of the labral brush "exhibits a peculiar striated appearance." Christopher's term for these rods , "cross bars, " is used here. On each lateral labral brush of A. fitchii larvae between forty- five and fifty of these bars are present and each bears approximately twenty hairs. Thus each lateral brush contains nearly a thousand hairs. A similar number of hairs is pres ent in each lateral brush of C. inomata larvae.

The cross bars are cuticular thickenings of the tessellated mem- brane (fig, 5) with their dorsal ends free in this membrane next to the dorsal sclerite of the labrum. When the cross bars are torn away from the tes sellated membrane and the hairs, depressions on them where the

Fig. 3. Ventral view of the labrum of the larva of Aedes fitchii with the lateral labral brushes extended. Numbers indicate hair types.

Fig. 4. Details of labral hair attachments of the larva of Culex territans .

53

Fig. 5. (a) Forked bases of labral hairs of Aedes larvae; anterior views, (b) The

relationship between hair base, cross bar, and the tessellated membrane, and the holes and depressions left in this by the removal of hairs and cross bars. Open stipple stretchable cuticle (stains blue); close stipple, flexible but relatively non- stretchable cuticle (stains red). (c) Diagram showing how the hairs are brought together by the increasing angle of movement at greater distances from the brush sclerite, because of differential stretching between the cross bars and the tessellated membrane.

54

Mouthparts of Mosquito Larvae

hairs were attached can be seen. The other end of each cross bar is curved into a hook; it terminates in t-he brush sclerite which is roughly triangular and is attached to the median part of the torma by an apodeme (fig. 3). Muscles that move this sclerite are inserted on the posterior tormal apodeme (fig.l). When the hairs are pulled off the membrane, their forked bases, the cross bar s, and part of the membrane comes with them. This leaves holes in the membrane and confirms that the cross bar s are more strongly attached to the hair bases than to the membrane. The hole may be rhomboid, square, pentagonal, hexagonal, oval, or roughly circular and form a mosaic pattern on the membrane which gives it its names. The cross bar s leave depressions in the tessellated membrane.

When this complex is stained with Mallory's the cross bars and the hair bases stain red indicating rigidity, while the tessellated membrane and small parts of the hairs above their bases stain blue , indicating str etchability. The edges of the holes may be outlined in red perhaps because of some change in the character of the material of the membrane resulting from tearing.

The ends of the epipharyngeal bar are attached to the posterior parts of both tormae (figs. 1, 3). At the anterior end of each torma a narrow sclerite projects medially. These sclerites are known as trans- verse bars (Shalaby 1957a) or palatal bars (Christophers I960). Their structure in A. fitchii is slightly different from that in A. aegypti as described by the above authors. The bars of A. aegypti are sleudof and from each a small curved sclerite projects anteriorly. In A. fitchii they are stout and curved medially, and are attached by thin sclerites to the tormae. In Culex territans the bars are straight and have wide basal parts.

In the species examined only. the posterior apices of the tormae stain blue; the remainder of these structures with their apodemes retain their brown color . Thus the, tormae and their apodemes a,re rigid, highly sclerotized structures. The associated membranes stain light blue.

The labrum of the predatory Chaoborus americanus larva is greatly reduced; it lacks brushes but possesses a few short stiff bristles at the tip of the labral sclerite (Cook 1956). These bristles stain dark red.

The Epipharynx and Preora! Cavity

The epipharyngeal apparatus lies between the posterior ends of the tormae and combs food particles from brushes to the mandibles. Schremmer (1949) called it the "Epipharynx - appar at" because it is musculated and has an active rather than a passive function.

The structure of the epipharynx in the species examined is very similar to that described by Shalaby (1957) and Christophers (I960) in A. aegypti , In A. fitchii andthe other browsers the hairs are coarser than in Culiseta morsitans and Culex territans , The spines and hairs stain dark red in A. fitchii which indicates medium hardnes s ; they stain lighter redin C. morsitans and C. territans and are probably softer in thes e species , The epipharyngeal bar stains medium blue in all specimens. That this flexible structure can move anteriorly and posteriorly has been observed in living larvae of A. aegypti and C. territans

Pucat

55

The post- epipharyngeal area consists of a membrane between the epipharynx and the pharynx. It is similar to that described by Cook (1944b) in Theobaldia incidens {-■ Culiseta incidens ). Two pairs of muscle strands originate on the frontoclypeus , one of these forks before its insertion in the membrane between the epipharynx and the pharynx. Since these muscle strands have a common origin on the cranium med- ially of the antenna (fig 1), I consider them as fascicles of one muscle, the postepipharyngeal.

The Mandibles

The mandibles of mature /let/es fikAii larvae consist of flattened, roughly quadrilateral lobes with their mesal ends produced into strongly sclerotized toothed processes and lower seta-bearing lobes. They are similar to the mandibles of most culicine larvae which have been des- cribed by other authors.

On the mesal margin of each mandible is found a fringe of pig- mented, long,mesally directed setae with stout bases and sharp points. Shalaby ( 1 957a) called this fringe the mandibular comb when he described itin A. aegypti , The number of the curved, stout and sharply pointed s etae varies in fourth instar larvae of the species that I examined. Eleven were usually found in A. fitchii , nine in C. inornata , and fifteen in A. aegypti , Another series of setae extends meso-dor sally from the dorsal side of the mandible, medially of the large lateral bristles; this series Shalaby names the mandibular brush. In C. inornata it usually consists of 40 setae; in A. fitchii of 54. The number of lateral bristles is variable between species, but constant in all the species seen; in A. fitchii two are present and in C. inornata three. When the mandibular brush and comb setae of the Aedes and the Culiseta browsing species are stainedwith Mallory’s their bases stain blue, and thus are soft; the re- maining parts stain dark red, and are harder. The mandibular setae of the filter -feeding species , Culiseta morsitans and Culex territans are softer than those of the browsing species. The lateral bristles remain brown in all the species examined. All the mandibular bristles and setae in the mandible of Chaoborus americanus stain dark red or remain brown.

The number of teeth in A. aegypti , as described by Shalaby, is similar to that in A. fitchii and to the other Aedes species that were examined. The number of ventral teeth in C. inornata is similar to that

found in the browsing Aedes species, but dorsally only three teeth are present in C. inornata whereas five are present in all specimens of all the Aedes species. The extent of heavy scler otization in the tips of the man- dibles, mainly the teeth, is approximately the same in C. inornata and the browsing Aedes species. The heavily sclerotized area is smaller in the filter feeders, and it is largely extended in the predatory Chaoborus americanus and Mochlonyx velutinus . These characteristics agree with the characteristics of browsers, filter feeders, and predators that Surtees ( 1 959) discus ses . Medially, on the dorsoventral ridge of the mandible a group of long spines reachesthe anterior part of the pharynx. Schr emmer (1949) discusses the function of similar spines on the mandible of Anopheles maculipennis ■ Anterior and po sterior mandibular articulations are indicated in fig. 1.

56

Mouthparts of Mosquito Larvae

The Maxillae

Each maxilla of A. fitchii (fig. 7) consists of a rectangular flattened lobe which bear s a brush of long hair s apically, and a series of three rows of short hair s medially in an area demarcated by a suture on the oral (dor- sal) side. Proximal to the palpus is a triangular sclerite about half the width of the main looe, which is attached to the s e structures and to the post gena by a membrane. This sclerite bears a spine medially. Baso-ventr- ally the maxillary palpus bears scler otized processes which articulate with a postgenal articular proces s inside the head (fig.l). The mandible also articulates with the postgena and the maxilla at this point. Two muscles are inserted in the center of the main maxillary lobe;a single strand originates on the subgena mesally to the posterior tentorial pit, and a double strand originates on the postgena posterior to the eye (fig. 1),

To decide what parts of the maxilla of A. fitchii larvae are homo- logous with parts of maxilla of other insects, the r elation between scler- ites and musculature must be considered. It is generally accepted that as Imms (1944) states . . the Mecoptera are the nearest living repre- sentatives of ancestors of Diptera. . , " This view is also expressed by Applegarth ( 1 939) , Ferris and R ees ( 1 939) , Potter ( 1 948) , Hinton ( 1 958) , and other s . We should therefore lookfor homologies of the maxilla of the mosquito larva in the Mecoptera and in other members of the suborder Nematocera. The palpus is the only structure on the homology of which all the authors agree. Since the palpus is connected to the base of the main maxillary lobe, and since the palpus in all insects is connected to the stipes , it seems logical to consider this lobe as the stipes. According toSnodgrass (1936) and Das (1937) the stipes can be distinguished by the origin of the muscles of the palpus. However, this criterion does not apply when the palpal muscles are absent as from mosquito larvae and larvae of Tipula and Bibio as described by Das (1937) and Cook (1944a), The two muscles that are present in this structure are probably the cranial flexors of the stipes (rather than of the lacinia). The double strand which originates on the postgena is one of these, and the adductor of the stipes which usually or iginates on the tentorium is the other. In the culicid larva the origin of the latter has shifted to the subgena.

Snodgrass (1935) and Das (1937) hold that the lacinia has a cranial flexor and the galea has only a stipital flexor in larval and adult stages of many insects . Das also states that many larvae lack the flexor of the galea, but when the lacinia is present its cranial flexor is always retained. The same author adds that the cranial flexor of the lacinia plays an important role in the interpretation of the lobes. No trace of stipital flexor was foundin any culicid larva examined. The only cranial flexor present is inserted so close to the median side of the main lobe that it is almost on the bri stle- cover ed area which is demarcated by a suture on the oral side of the lobe (fig. 7), Furthermore, this median bristly area functions as a lacinia. Therefore I agree with Shalaby (1957a, 1958) that this part of the maxilla is the lacinia, and that the cranial flexor of the lacinia now functions as a stipital flexor.

In the larvae of Panorpa both galea and lacinia are present (Das 1 937) ;

in Apterobittacus only the lacinia i s present in the larval stage and the galea appear s in the pupal stage (Applegarth 1939); in both Tipula (Dasl937)and Bibio (Cook 1944b) only the lacinia is present in the larval stage. The

57

mandibular brush’

mandibular comb

dorsal teeth

0. 5 mm

brush of dististipes

disti stipe s

Fig. 6. Ventral view of the left mandible of mature larva of Aedes fitchii Fig. 7. Dorsal view of the left maxilla of mature larva of Aedes fitchii .

58

Mouthparts of Mosquito Larvae

triangular sclerite which is considered as the palpifer by most authors I believe to be at least a partial vestige of the cardo. In the larva of Panorpa the car do has a relative size, shape and position similar to that in the mosquito larva, and it also lacks musculature (Das 1937) . In the lar-

vae of each of Apterobittacus , Bibio . and Tipula species the structure named as cardo by the respective author s , is proportionately larger than in the larvae of Aedes , Culex , and Culiseta. In the former three larvae the so- called cardo extends posterior to the stipes and the palp. If this structure is homologous with the triangular sclerite in the mosquito larva then this sclerite must be the cardo and not the palpifer. However Hinton (1958) points out that the stipes is divided in to a basistipes and dististipes in all the Panorpoidea except the more specialized Diptera. The same author further states that failure to recognize the fact that the stipes is sub- divided in primitive forms of all recent orders of the Panorpoidea has resulted in the misidentification of the dististipes as the palpifer. Hinton also states: "in the Panorpoidea in which the cardo has become fused to the basistipes the combined structure which may be called the cardostipes has almost without exception been identified as the cardo and the disti- stipes as the stipes. For instance, the cardo plus basistipes of Bibio is called the cardo and the dististipes is called the stipes by Imms (1944) and Cook (1949). . . " In the light of Hinton's statements then I consider the triangular sclerite of the mosquito larval maxilla as homologous with the fused cardo and basistipes. The main maxillary lobe is the disti- stipes plus the lacinia. In addition Hinton mentions that within the Nematocera a fusion of the cardostipes with the dististipes takes place for example in the Culicidae, but he does not specify in vdiat group of the Culicidae. He may be referring to the genus Anopheles , for in that genus there is no triangular sclerite proximal to the maxillary palp and the dististipes as in the genera Aedes, Culex, and

Essentially the same structural arrangement of the maxilla was found in all the Aedes , Culex , and some Culiseta larvae that I examined. Some difference from the browsers was found in the shape of the maxillae of Culex territans , Culiseta morsitans , Aedes canadensis , and A. cinereus Each maxilla in these species is cone- shaped, wide at the

base and narrow at the apex where a brush of simple hairs is attached. The maxillae of most browsers are similar in shape to those of Aedes fitchii . Between the browsers and filter feeders differences occur in the number and length of hairs on the distal end of the dististipes and on the lacinia. In the maxillae of both filter feeder s and browser s the apical brush hairs of the dististipes are longer than the lateral hairs of the lacinia, and in the filter feeders all these hairs are proportionately longer than in the browser s . The longest maxillary hairs in Culex territans and Culiseta morsitans are approximately one and a half times as long as the dististipes; whereas the homologous hairs in A. fitchii and the other Aedes browsers are only approximately as long as the dististipes, and in both Culiseta inornata and C. impatiens (Walker) they are half the length of the dististipes. The maxillary brushes of the browsing Aedes species are composed of more hairs than those of the filter feeding species. The maxillae of C. inornata and C. impatiens larvae have brushes consisting of very few hairs, thus resembling the maxillae of predatory larvae.

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Another similarity of the maxillae of these two Culiseta species to the predatory larval maxillae is the fusion of the palps with the cardobasi- stipites.

With Mallory's stain the bases of the maxillary brush hairs of browsing larvae stain blue and the remaining parts red, but the whole hairs stainblue in filter feeders. Thus the maxillary bru shes of browsers are stiff, a feature of obvious value in their activity.

The short medial bristles of the lacinia are arranged in three rows in all the species that I studied; they are more numerous in browser s than in filter feeder s. These hairs are longer in A. fitchii and the other Aedes browsers than in C. inornata and C. impatiens . In all the browsers these hairs stain red, indicating moderate stiffness. The hairs of the lacinia of the filter feeders stain blue and thus are soft.

The Labium and Hypopharynx

I consider the labium of the larva of A. fitchii to consist of the prementumand the submentum. This view is in agreement with Cook's (1944b) interpretation for other genera. The prementum (fig. 2) is a rectangular membranous area bearing a series of serrated sclerites and papillae, and is situated between the hypopharynx and the mouth opening dor sally, and the triangular serrated submental plate ventrally.

Dorso-ventrally two long sclerites extend through the centre of the prementum and dorsally terminate ventral to six small serrated sclerites which project ventrally from the membranous base. On the sides of the membrane three serrated plates are situated ventrally. These three plates are connected to each other, and dorsally to the small central serrated sclerites. Each plate has a different number of serrations, which vary in different species. In A. fitchii . the dorsal plate has four serrations, the median plate nine, and the ventral plate five. Six larvae of each of two closely related species, Aedes hexodontus and A. pun c tor were also examined, and the aver age number s of serrations were found to be; dorsal plate 5 serrations in A. hexodontus , 4 in

A. punctor ; median plate 6 in A. hexodontus , 9 in A. punctor ; ventral plate 6 in A. hexodontus , 10 in A. punctor . This may be a useful taxonomic char- acter for separating closely related species. Considerable car e is required in preparing the slides if the serrated plates are to be seen clearly.

Since these plates in all the species of Aedes , Culiseta , and Culex, that were examined stain light red basally and dark red to orange distally, they are quite hard. This is understandable because the mandibular teeth which are of similar hardness strike against them. The hardness of both structures could be felt with dissecting needles. In the Aedes species a group of broad, apically serrated hairs originates on the mid- ventral side of the premental lobe. Broad, but not serrated hairs occur in the same position in the Culiseta and Culex species. These hairs are numerous in Aedes and Culiseta but very scarce in Culex. In all the species examined they stained medium red with Mallory's.

On the premental lobe laterally, between the central and the lateral serrated plates four small papillae are present on each side in all the species of Aedes , Culiseta , and Culex that I examined. The most

60

Mouthparts of Mosquito Larvae

posterior papillae are double on each side; the more anterior two arise singly. Two similar papilla-like processes are present in the membrane dor sally between the serrated plates and the salivary duct opening. In all the species considered the papillary structures stained red, and the basal membranes light blue. In feeding larvae, food often collected in the spaces between the papillae and the serrated plates.

It is difficult to homologize the structures of the labium because of its degenerate nature, but since a pair of muscles attaches the rectangular lobe to the subgena medially to the posterior tentorial pits (fig. 1), these muscles are considered as the premental muscles by Cook (1944b, 1949), Snodgrass (1959), and others, Snodgrass

refers to the lobe as the labial plate. I agree with Cook in calling it the prementum.

The premental membrane is dorsally suspended from the hypo- pharyngeal bars. A weak suture continues between these bars and dorsally of the premental membrane, thus demarcating an oval membranous hypopharyngeal area above the prementum. The opening of the salivary duct is located between the premental and hypopharyngeal lobes. This was so in all the species examined including A. aegypti although Christophers (I960) shows it in the center of the prementum.

The triangular serrated sclerite below the prementum has been variously named (table 1). I agree with Cook (1944b, 1949) that it represents the submentum. Salem (1931) considered it homologous with the submentum, but thought that the customary name, mentum, should be retained. Snodgrass (1959) believed it to be an extension of the sub- gena. Jones (I960), following Snodgrass, calls it the hypostomium in the larvaof Anopheles quadrimaculatus . My main r eas on for disagr eeing is that in all the species examined this sclerite articulates with the subgena, and therefore is unlikely to be an extension of it. Generally the submentum of insects articulates with the ventral part of the cranium (Snodgrass 1933) . Snodgrass (1959) however, does not mention that this triangular structure arcticulates with the subgena. He states that it is continuous with the subgena, as in the head of Chironomus described by Grouin (1959) who calls it the hypochilum. Miall and Hammond (1891) indicate that this plate in Chironomus seems to correspond to the submentum of orthopterous insects.

The submentum stains orange basally with Mallory's and remains dark brown apically in all the Aedes , Culex , and Culiseta larvae I examined. It is thus a very hard structure. In the species examined the number of serrations on it in mature larvae is usually constant; data are given in table 2.

The lightly sclei otized fringe of hair s (figs. 1, 2) attached to the submentum ventrally stains similarly; I consider it a part of the sub- mentum since it is very intimately connected with this structure. Cook (1944b) calls it the aulaeum.

The Pharynx

The structure and musculature of the pharynx of A. fitchii and C. inomata larvae are similar to those of Theobaldia incidens [- Culiseta

incidens ) described by Cook (1944b). The large dor sal and vent- ral sclerites stain light orange in all the Aedes , Culex , and Culiseta larvae

Mouthparts of Mosquito Larvae

61

TABLE 2 - The numbers of serrations on the submentum of the larvae of mosquito species.

Species	No. of submental serrations	Species	No. of submental serrations
Aedes spp.			Aedes spp.
campestris	27. 0	(D*	sticticus	21.6+0. 5	(3)
canadens is	20. 0	(1)	stimulans	28. 0	(1)
cinereus	25. 0	(1)	vexans	26. 0+0. 7	(5)
excrucians	20. 5	(2)	Culiseta spp.
fitchii	20. 6±0. 9	(2 0)	impatiens	25. 0	(1)
hexodontus	24. 6±1. 1	(5)	incidens |	18. 0	(1)
implicatus	18. 0	(1)	inornata	23.9+2.2	(17)
increpitus	25. 0	(1)	morsitans	19. 0	(2)
impiger	20. 5± 1 . 3	(4) |	Culex Spp.
pionips	24. 0	(2)	pipiens	21. 0	(2)
punctor	27. 1±0. 7	(6)	tarsalis	13. 0	(2)
riparius	23. 0±0. 7	(5)	territans	13. 0	(2)

* average ± S. D. of the mean (where applicable); number of specimens examined in parentheses.

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Mouthparts of Mosquito Larvae

examined. The lateral dor sal hair s stain light red, and the inner filtering hairs stain light blue in most species. Schremmer (1949) described the filtering function of the pharyngeal hairs in Anopheles maculipennis larva.

Discussion

It is difficult to decide on the homologies of degenerate structures like t h e maxilla and labium of mosquito larvae. Shalaby's(1957d) interpretation of the triangular labial sclerite as the paraglossa, and the aulaeum as the glossa is unique, and seems unreasonable. The areas which I consider as the hypopharynx and the prementum Shalaby regards as the hypopharynx. Medio- laterally on the premental lobe a pair of muscles is inserted. These muscles originate on the ventral sclerite of the head which Shalaby considers as the submentum and which I regard as the subgena. It is difficult to agree with Shalaby 1 s interpretation of the labium and the hypopharynx for the following reasons: firstly,

as far as is known, the hypopharynx in insects is not connected with the paraglos sa, but in the mosquito larva, in Shalaby' s interpretation the " hypopharynx " is firmly attached to the "paraglossa". Secondly, other authorities on the morphology of insect larvae (Cook 1944, 1949; Hinton 1958) state that the retractor muscles of the hypopharynx are absent in Diptera. Thirdly, when the retractors of the hypopharynx are present they arise on the postoccipital ridge in the Trichoptera, and on the tentorial bridge in the Lepidoptera (Hinton 1958), but not on the "submentum" where these muscles originate in the mosquito larva according to Shalaby' s interpretation.

Very few muscles which could serve as guides to homology are present, and this is partly why disagreements exist among the various morphologists who have studied mosquito larval mouthparts.

Ferris ( 1 948) postulates the following principle: " the evolutionary

changes ,are first to be accounted for by modifications of pre-existing

structures , or by loss of pre-existing structures; Only after these

possibilities have been exhausted will we assume that a completely new structure has been developed. ..." This principle can be applied to mosquito larvae and to the larvae of other primitive Nematocera when we compare them with panorpoid larvae. In mosquito larvae noticeable modification from Panorpa is seen in the labrum and in the mandibular teeth. Losses and fusions of pre-existing structures are evident in the mosquito larval maxilla and the labium.

A difference was found in the hardness and flexibility of the cuticle of the mouthparts of the filter feeding, browsing, and predatory mosquito larvae. Essentially, the mouthparts of the filter feeders are rather soft except for the labral brush hairs and the mandibular teeth; the mouthparts of the browsers are harder, and the mouthparts of the predatory larvae are the hardest of all, especially the mandibles, which are highly sclerotized.

The tips of the simple labral brush hairs of the filter feeding and browsing larvae are softer than the main parts of the hairs. The labral brush hairs of these groups of larvae are much harder than they appear to be since they are refractory to stain until after boiling in a relatively strong (8%) solution of KOH. It was interesting to find that

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63

the serrated ends of the lateral labral brush hairs of the browsing larvae stain blue and thus are soft combs rather than hard ones as they might be expected to be when their function is considered. Since they are soft it is probable that when they rub over surfaces soft particles are detached and then dir ected towards the mouth. The physical characteristics of the cuticle were estimatedby manipulating the mouthparts, and the impres- sions obtained agreed with the indications from staining.

The serial row attachment of the labral brush hairs to their respective bar s is similar in the browsing and the filter feeding larvae. Christophers (I960) also noted that the hair attachment is similar in the larvae of a Culex species and of A. aegypti .

In table 3 it is indicated that a reduction occurs in the numbers of hairs or bristles on the various mouthparts from the filter feeders to the predators. In the same series an increase in the scler otization of the mandibular teeth is evident.

TABLE 3 - Similarities and differences in the mouthparts of filter feeding, browsing, and predatory mosquito larvae.

	Labral brush hairs | i	Maxillary hair s	Premental hairs SclerotizE	J Mandible ition Plane of action
Filter
feeders
C. morsitans	many long	many	few	slight
	thin simple	very long	short moderate
Intermed.
A edes	many thin	very many	many	heavy
cinereus	simple	long	long moderate	nearly
				dorso-
Brows er s				ventr al
Aedes fitchii	many thick	very many	many	heavy
	serrated	long	long moderate
Culiseta	many thick	few	many	heavy
inornata	serrated	short	long moderate
Predator s
Mochlonyx	few short	very few	many	very
velutinus	thick	very short	mostly	heavy 1 antero-*
	serrated		long slight	lateral
Chaoborus	very few	none	very	very
americanus	thick short		few	heavy
	serrated		short none

It is interesting to note that the same genus is represented by filter feeding ( Culiseta morsitans ) and browsing larvae ( C. inornata and C. impatiens ) whose mouthparts tend towards the predatory type. Most

64

Mouthparts of Mosquito Larvae

of the Aedes species that were studied are browsers, but the larvae of Aedes cinereus Meig. and A. canadensis lack serrations on their labral brushes, have more weakly sclerotized mandibular teeth than the other Aedes species, and their maxillae are similar to those of the filter feeders. Thus morphologically these species seem to be intermediate between the filter feeders and the browsers.

From table 3 it is also evident that the plane of action of the mandibles in the predatory larvae tends towards that of the longitudinal axis of the body which is a character common both among the larvae of the higher flies, according to Cook (1949), and among predators generally.

FUNCTION OF THE MOUTHPARTS OF MOSQUITO LARVAE

Procedures

The movements of the mouthparts of mosquito larvae and actions resulting from thes e movements were studiedintwo situations: behaviour of larvae (mostly Aedes ) was observed in the muskeg pools in the Flatbush area (100 miles north of Edmonton) in the summers of I960 and 1961; more extensive observations were made on active larvae in artificial containers in the laboratory.

After being collected the larvae were kept in pint glass jars, and in order to retard their development when not being observed they were kept in the refrigerator at 40°F. The larvae were observed in groups and individually in the glass jars and some details of movements of their mouthparts were seen with the aid of a 1 OX hand lens. Individual larvae were placed in small vials and their mouthparts were observed from the side with a hand lens. A viscous solution of an inert material such as methyl cellulose was also used to slow down the motions of the mouthparts so that details of their actions could be studied.

Larvae of A. aegypti and Culiseta inornata reared in the laboratory were observed. Other species of Aedes and Culiseta were collected in the areas of Flatbush, Edmonton, Lake Hastings , Banff, and Seebe, Alberta. The larvae were identified with the keys of Rempel (1953) and Carpenter and La Casse (1955).

Since the mouthparts are ventral it was desirable to observe larvae from the ventral side; three methods were used for this. For all the methods a container was made by cutting a 1 in long piece of aplastic vial of 1 in diameter, and gluing it to a microscope slide which formed the bottom. The container was filled with either pond water or distilled water and food was added. Usually one larva was studied at a time, but sometimes two were observed in the same dish.

By means of two concave mirrors, light from two microscope lamps was directed on the larva through the bottom of the container. An image of the ventral surface of the larva was reflected by two plane mirrors at 45°, one below the container and one below the objective of a stereo-binocular microscope. A satisfactory view of the mouthpart

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movements was obtained in this apparatus. The movements were most clearly seen at magnifications of six or twelve diameters. More detail was seen under 25X and 50X, but the images were blurred, especially at 5 OX.

A second method of observing the mouthparts was by turning the body and eyepiece of the binocular microscope upside down and focussing on the larva above the microscope. The best image was obtained by this method which was used most often. Fluorescent light from above and tocussed light from below were used separately and in combination.

A third and most convenient method of observing the movements of larval mouthparts was through a metallurgical binocular microscope with the stage above the objective lens. In this method it was possible to have the light coming only from above.

Particles of activated charcoal or methyl red were placed in the containers with the larvae to show the directions of the currents set up by the mouthparts.

Observation of the Mouthparts in Action

The operation of the lateral labral brushes was studied by direct observation of living larvae and by manipulation of prepared material. The mechanism of action in each type of mouthpart is described sep- arately below.

Browsers

In this group contraction of the labral muscles exerts tension on the brush sclerite which in turn pulls the tessellated membrane and the cross bar s by their hooks. This causes the hairs of the brush to move ventr o-medially. The hairs spring back outwardly through the elasticity of the tessellated membrane. The inward and outward move- ment of the hairs is thus caused by the differential elasticity of the tessellated membrane and the cross bars. The bases of the hairs are connected with the cross bars, and forjc on either side of them (fig. 5). The bifurcations are short, and their ends terminate in the tessellated membrane belowthe cross bars. The stretch of the tessellated membrane allows the part of the hair which is attached to the rigid cross bar to move more than the tips of the fork, so that the hair pivots about this attach- ment to the cross bar, and its tip swings ventr o-medially. Relaxation of the labral muscles allows the hair s to return to their original positions through the elasticity of the tessellated membrane.

The angle through which a hair swings should increase with its distance from the brush sclerite since it is separated from this by a greater length of the elastic membrane. This would have the effect of bunching the hairs together in the median position and allowing them to fan out in the lateral position, which was repeatedly observed to happen.

The main feeding current, produced by the lateral labral brushes, is directed toward the epipharynx and the mouth by the median labral brushes. When creating a current the lateral labral brushes vibrate from

66

Mouthparts of Mosquito Larvae

TABLE 4 - Mean frequency and duration of movements of the lateral labral brushes of larvae over one minute periods at 24 to 27°C.

	4th instar	2nd and 3rd instars	4th instar
	means	of 3 larvae	means	of 4 larvae	means of 3 larvae
Time	Cycles	Average	Cycles	Average	Cycles	Average
in	per	duration	per	duration	per	duration
min.	sec.	sec.	sec.	sec.	sec.	sec.
5 th	1.7	28. 5	2. 6	11. 0	2. 0	30. 0
10th	2. 7	17.7	3. 0	11. 0	3. 3	13. 0
15th	3. 3	37. 0	2.4	62. 5	1.8	8. 0
20th	3. 1	35. 5	3. 1	24. 0	1.8	7. 0
25 th	3.4	65. 0	2. 8	16.4	1.8	7. 0
30th	3. 5	27. 0	2. 6	31. 0	1.8	4. 0
35 th	4. 0	25. 0	3. 0	11.7
40 th	3. 7	36.4	2. 8	12. 0
45th	3. 6	68. 0	2. 1	6. 0
50th	4. 5	32. 5	4.6	13. 7
55th	3. 7	61. 0	3. 2	12. 5
60th	3. 5	40. 3	3.4	16. 6

postero-medially to anter o-later ally. The brushes of A. aegypti may vibrate for as long as 2. 5 min without stopping. Then they usually stop for 5-10 sec before resuming. The more usual timing is vibration for 50 sec, stop for 5-10 sec. and then vibration again. In Culiseta inornata and in the browsing Aedes species the duration of movement is shorter. Frequency and duration of movements for C. inornata and A. aegypti and indicated in table 4. Table 5 shows activity of individual 4th instar C. inornata larvae, each of which was observed for 3 0 minutes. During each 30 minute period the activity of the whole body and of the mouthparts, was observed, and the percentage of time spent in each observable activity was calculated.

Feeding and locomotory activities of approximately 50 C. inornata larvae were observed individually for various periods of time throughout the period of the study, and many more were observed in group behaviour. Much similarity was noticed in the pattern of behaviour of the various individuals, and almost any larva could be chosen to represent the common sequence of activities. The following is a summary of the activities of a 4thinstar C. inornata actively browsing larva (no. 6 in table 5), observed for 20 min at a magnification of 25X. The container was filled with pond water.

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During the fir st minute the larva was stationary; it was suspended from the water- surface with labral brushes extended. For the next 10 sec the labral brushes, the maxillae, and the mandibles created a current, then a 10 sec period of rest followed with the mouthparts retracted and the whole body still. During the first 5 min period such a succession of currents in which all the mouthparts participated was produced four times, and each time the labral brushes moved about 15 times. The mandibles and the maxillary brushes also moved approx- imately as many times as the labral brushes.

TABLE 5 - Percentage of time spent by 4th instar C. inornata larvae in different activity states over a 30 min period.

Body	of larva	Labral brushes
Stationary	Moving	R etracted	Extended	Moving
48	52	37	48	15
52	48	18	59	23
62	38	22	57	21
44	56	0	63	37
53	47	6	25	69
12	88	35	29	36
45	55	5	16	79

The larva browsed on a filamentous piece of plant for ten seconds. The piece of plant was enclosed by the labral brushes and the mandibular teeth struck it. Then the larva moved to a chickweed leaf and browsed on its edges for 18 sec. The median hairs (type 3) of the lateral labral brushes held the edges of the tissue while the more lateral hairs (type 3) of the brushes produced a current which moved the larva forward along the leaf. The mandibular teeth struck the tissue. Then the tissue was left and further currents were produced by the mouth- parts. Pieces of debris passed into the current which was produced con- tinuously for approximately 20 sec. Mandibular teeth chopped off small pieces of decayed material, some of which went into the mouth and the remainder moved out with the current. Again a piece of plant tissue was browsed upon, and was then propelled posteriorly. When one end of the plant was at the submentum the aulaeum clung to it for a few seconds, but with the subsequent current the tis sue was for ced posteriorly and towards the bottom of the container.

During the next ten minutes continuous movements of the mouth- parts occurred 15 times, each time the duration of the current was approximately 15 sec.

The amount of brush movement and body movement varies among larvae of different ages and different species. Fourth instar larvae are more sluggish than younger ones, and 4th instar Culis eta inornata and Aedes fitchii larvae are more sluggish than the corresponding instars

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Mouthparts of Mosquito Larvae

of A. aegypti (table 4) . Shannon (1931) and Christopher s (I960) also noticed that A. aegypti larvae moved considerably faster than the larvae of most other species of mosquitoes. Fourth instar A. aegypti larvae can ingest char coal particles faster than 4th instar C. inornata larvae. When activated char coal was placed in a container with 3 A. aegypti larvae and in another container with 3 C. inornata larvae (all 4th instar), the guts of the former were filled in from 90 to 105 min, whereas the guts of the latter species were only filled after 3.5 hr. Larvae of all the species observed moved faster and more frequently when they were stimulated to activity by other organisms ( Daphnia , Cyclops etc. ).

When the brushes are not rhythmically beating to create a feeding or a locomotory cur rent they remain extended and separated into rows of four or five layer s (fig. 7) , or they are retracted (fig. 7). Particles which have been brought close to the mouth by the current continue streaming towards the mouth through the spaces between the rows of hairs, or if the brushes are retracted the particles come to rest on the maxillary brushes. If these are extended the particles stream into the mouth and some settle on the hairs of the pharynx, the mandibles , the maxillae and the prementum. The separation of the labral brush hairs into several rows (fig. 7) is possible because of the basal structure of the brush. Each row of hairs can be moved about the axis of its cross bar. Several rows can move in one direction together, and thus water can flow through the spaces between these groups of hair s. It also seems that the water currents can for ce the labral brushes to close. The muscles that insert on the tormal apodemes (fig. 3) extend the brushes by contraction. Relaxation of the labral muscles allows the hair s to return to their original positions through the elasticity of the tessellated membrane. This can be demon- strated in preserved specimens. The contraction of these muscles and of the epipharyngeal muscles was observed in living larvae of a filter feeder, Culiseta morsitans .

The feeding currents of Aedes and Culiseta browsers are fast and can carry large as well as small particles. Objects about one third the size of a larval head can be circulated in the stream (fig. 8), the current and the particles reach as far posteriorly as the fourth and fifth abdominal segments and extend about the same distance in front of the larva. Such circulation of particles can be observed when the larva is suspended in water and also when it lies on its dorsal side in an observation cell.

When a larva feeds just above a loose sediment (fig. 8) or browses its way forward through debris in a container, the particles that do not enter the mouth fall to the bottom of the container or cling to the brushes; they do not return to the feeding current. The feeding current is effective only in front of the larva, and it is slowed down behind the larval head. The water flows ventrally rather than posteriorly below the body of the larva. When the larva leaves the browsing area many particles remain on its labral and maxillary brushes , since what does not fall to the bottom of the container sticks to the brushes . Some filtering is done by the labral brushes , especially by the median serrated ends of the lateral labral brush hairs, quite large particles are found clinging to them. Since particles only slightly smaller than these have been found in the pharynx and in the intestine, and since most food seems to come into the mouth via the

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labral brush current, it seems reasonable to assume that the particles which pas sed into the mouth and eventually into the gut were filtered out by the brushes. The serrated brush hairs are useful in browsing, for as they move along surfaces they detach particles from them, many of which are consumed.

With its labral brushes a browsing larva can attach itself to a grass stem, to the side of a container, or to a body of a pupa or another larva. While the labral brushes cling to surfaces the maxillary brushes produce a current. The browser 's maxillary brushes can create currents that are as strong as those of the labral brushes. This was observed in fourth instar larvae of the following species:4ec/es cataphyllaT)yB.r ,A. sticticus[NLeigen), A. communis (De Geer), A. fitchii ,A. punctor ,A. riparius,A. canadensis , Culiseta inornata and C. impatiens . The larvae can also browse on parts of their own body, especially on the posterior regions of the abdomen. This was observed particularly in containers where Aedes and Culiseta larvae were crowded. Many times larvae, especially C. inornata and Aedes canadensis ., were seen browsing on the tips of their own abdomens and creating currents at the same time. They were in loop-like positions and moved in circulating paths of the water surface. This was particularly notice- able in the laboratory with the larvae of A. canadensis ; on one occasion in June I960, 20 to 30 larvae turned in this manner for several minutes,

individual larvae turning for as long as five to six minutes. Christophers (I960) states that larvae browse on parts of their own bodies, especially on the posterior parts, when they are starving. My observations agree, for in situations where this behaviour took place little food was present.

Interfacial feeding (Renn 1941, and fig. 8) is a common method of feeding in the Anopheles filter feeding larvae. Third and fourth instar C. inornata , A. aegypti , A. fitchii , A. punctor , and A. riparius larvae also brow- sed at the water surface without browsing on their siphons at the same tim'e. In this second type of filter feeding only the head of the larva was at the water surface and the rest of the body remained under water .

In most browsing activities all or most of the mouthparts are employed. When an object such as a long thin piece of decaying grass comes into the feeding current, it comes in contact with the mouthparts as follows :firstly, the serrated lateral labral brush hairs (median type 3) hold a part of it, and push the remainder posteriorly; second, it slides over the central labral brush; third, it passes between the epipharyngeal bristles; fourth, the mandibular denticles strike it as it passes by, and if a small piece of it is thus torn off it may go posteriorly with the current, it may be drawn into the mouth, or it may settle on the prementum; fifth, it passes between the maxillary brushes; sixth and finally, the particle of grass touches the submentum and the aulaeum. During this process some of the median labral brush hairs hold the particle while the remaining hair s of the brush produce currents.

Sometimes parts of the lateral labral brushes move only slightly (median type 3 hairs) whereas the hairs of their most posterior (types 2 and 3) move more actively. More commonly, all the hairs on the brushes move simultaneously when producing a current. When a larva comes to a stop after moving about in a container , it will gradually extend

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surface view of currents

Fig. 8. Movements of labral brush currents of browsing larvae; (a) interfacial sur- face feeding current, (b) current produced under the water surface, (c) current used to stir up debris from the bottom.

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or contract the brushes.

Most of the observations on the coordination of moving mouthparts were on Aedes aegypti and A. fitchii larvae which had been slowed down in a