14 THE CANADIAN ENTOMOLOGIST January 1981

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1 A SIMPLE CHARACER FOR RECOGNIZING SECOND AND HIRD INSAR LARVAE O FIVE CANADIAN MOSQUIO GENERA (DIPERA: CULICIDAE) ROGER SAVIGNAC and ALAIN MAIRE Groupe de recherche sur les insectes piqueurs, Universit6 du Qu6bec rois-rivi res G9A 5H7 Abstract Can. Ent. 113:13-20 (1981)) Second and third instar mosquito larvae of 34 Canadian spe"ems.belonging-eo-t "genera Anopheles,,'ledes, Culex, Culiseta, and Wyeomyia may be reliably and simply separated by the absence or presence of the thoracic setae, 8-M and 7-, and even more so by the number of the metathoracic setal support plates. As the 7- seta is always inserted on a plate, a larva in the third stage will bear one more plate than in the second stage. In the species studied, the number of metathoracic plates is constant for a given genus at a given instar. hus an intermediate instar larva, once identified to genus, can be assigned to second or third instar fairly readily by counting the number of setal plates on one side of the metathorax. R6sum6 L'absence ou la pr6sence des soies thoraciques, 8-M et 7-, permet de diff6rencier sans difficult6 les deuxism et troisi me stades larvaires de 34 esp ces canadiennes de moustiques repr6sentant les genres Anopheles, Aedes, Culex, Culiseta et Wyeomyia. Les plaques sc16rotis6es t la base des soies permettent de les distinguer encore plus facilement. La soie 7- s'ins6rant toujours sur une plaque sc16rotis6e, une larve de troisi me stade portera toujours de chaque c6t6 du m6tathorax une plaque de plus qu'au stade pr6c6dent. Chez les espsces 6tudi6es, le nombre de plaques est fix6 pour un genre donn6 un stade donn6 de sorte qu'une larve identif6e au genre peut imm6diatement 8tre class6e selon son stade par le nombre de plaques situ6es de chaque c6t6 du m6tathorax. Introduction In the Culicidae, the first instar larva is easily recognized by the presence of an egg-burster on the vertex of the head capsule and by the lack of the cratal seta, 4-X (Dodge 1966). In most North American species, the fourth instar larva is identified by chaetotaxy and by sclerotization of the tenth segment (Smith 1969a; Wood et al. 1979). On the other hand distinction between the second and third instars is more difficult. wo methods have been used. One method is based on linear measurements of the head capsule, the siphon, and the anal segment (e.g. Smith 1969b; Makiya 1969; Smith and Brust 1971; Danks and Corbet 1973; de Oliveira and Durand 1978; Reisen and Siddiqui 1979). his method has limitations resulting from intraspecific variation and is valid only after an adequate number of specimens of a given species have been measured and analyzed statistically. he second method makes use of chaetotaxic differences (Bohart and Washino 1957; Knight 1964; Eddleman 1967, 1968; Smith 1969a; Yan 1977). For instance Smith (1969a) noted that in the 25 species of Aedes recorded in New England, U.S.A., the second instar has only one tuft on each side of the metathorax whereas the third instar has two tufts. he third instar of Anopheles waikeri heobald also has an additional seta on the metathorax as well as one on the mesothorax which. are absent in the second instar (Hurlbut 1938). he same is true of the third instar of oxorhynchites (Lynchiella) rutilus septentrionalis (D. & K.), as shown by Dodge (1964), as well as of several culicine species (Belkin and McDonald 1956; MacKenzie 1972; Harrison and Rattanarithikul 1973). In the setal numbering system suggested by Knight and Laffoon (1971) these two setal acquisitions are 8-M and 7-, except in oxorhynchites, in which they are 7-M and 7-. herefore it seems that these'two thoracic setal acquisitions, whatever the setal numbering system, are a useful character to differentiate second and third instar larvae (Harrison and Rattanarithikul 1973).

2 14 HE CANADIAN ENOMOLOGIS January 1981 In this study, we have verified that a generalization of this character seems valid for 34 Canadian species belonging to 5 genera, thus providing a method to separate second from third instar larvae. Materials and Methods he second and third instar larvae of 34 of the 74 mosquito species recorded from Canada (Wood et al. 1979) were studied principally in collections made from across Quebec (able I) by the Groupe de Recherche sur les Insectes Piqueurs de l'universit6 du Qu6bec t rois-rivi res. Larvae of intermediate instars of each species were separated by head measurements of specimens coming from the same natural breeding sites (Fig. 1). In the case of Anopheles earlei, instars were determined by rearing in the laboratory. he descriptions are based on material preserved in 70% ethanol. First and second instars were examined at 100x with a stereomicroscope and the later instars at 40x. Specimens were prepared for drawing by clearing in NaOH and mounting in an aqueous solution on concavity slides, so the larvae were not crushed between the slide and the coverslip. All drawings were made with the aid of a camera lucida at 250x using a compound microscope. Results and Discussion In all larvae of the 34 species studied the 8-M and 7- setae were lacking in the second instar larva and were present in the third. In the third instar of Aedes, Culex and Culiseta, both 8-M and 7- are long, strongly branched and inserted on a support plate. In the third instar of Wyeomyia smithii, 8-M is single and minute and does not arise from a plate, while in Anopheles earlei, 8-M and 7- are single and plumose. hus, the presence of only one of these two setae allows diagnosis a third instar larva. he 7- is always inserted on a plate, so a third instar larva always bears one more plate than a second instar on each side of the metathorax. In Anopheles earlei, the presence of three metathoracic plates indicates a second instar and four plates a third instar (Fig. 2); in Wyeomyia smithii, two plates indicate a second instar and able I. List of the 34 species studied for larval instar separation Anopheles (Anopheles) earlei Vargas Aedes (Aedes) cinereus Meigen Aedes ( Aedirnorphus) vexans (Meigen) Aedes ( Ochlerotatus) abserratus (Felt & Young) atropalpus (Coquillett) aurif er (Coquillett) canadensis (heobald) c antator (Coquillett) comrnunis (DeGeer) decticus Howard, Dyar & Knab diantaeus Howard, Dyar & Knab dorsalis (Meigen) euedes Howard, Dyar & Knab excrucians (Walker) fitchii (Felt and Young) hexodontus Dyar implicatus Vockeroth intrudens Dyar Aedes (Ochlerotatus) pionips Dyar provocans (Walker) pullatus (Coquillett) punctor (Kirby) sticticus (Meigen) stirnulans (Walker) (Protomacleaya) triseriatus (Say) Culex (Culex) pipiens Linnaeus restuans heobald (Neoculex) territans Walker Culiseta (Climacura) melanura (Coquillett) ( Culicella) rnorsitans (heobald) (Culiseta) alaskaensis (Ludlow) irnpatiens (Walker) inornata (Williston) Wyeomyia (Wyeornyia) srnithii (Coquillett)

3 Volume 113 HE CANADIAN ENOMOLOGIS Aedes canadensis Culex territans z Culiseta melanura IC Wyeomyia smith# o. 2 0;4 o.6 o e HEAD CAPSULE WIDH(mm) FIG. 1. Size distribution in head capsule width of larvae of Aedes canadensis, Culex territans, Culiseta melanura, and Wyeomyia smithii. Class intervals, 0.01 mm for 1st and 2nd instar larvae and mm for 3rd and 4th instar larvae. three a third instar (Fig. 3) while in Aedes, Culex and Culiseta, there is only one plate in the second instar and two in the third (Figs. 4-6). Examination of the intermediate stages on the drawings of Hurlbut (1938), Smith (1969a), MacKenzie (1972) and those of Harrison and Rattanarithikul (1973) indicates that the number of metathoracic plates seems constant in a given instar of a given genus, so intermediate instar larvae identified to genus (see Dodge 1963) can be assigned to instar fairly readily by counting the metathoracic plates which form around the bases of setae (able II). However a more complete study, especially

4 16 HE CANADIAN ENOMOLOGIS Anopheles earle/ Janu 1981 P M 3rd INSAR !,I P 2nd INSAR /, - o 3 I- I FIG. 2. Left lateral view of thorax of Anopheles earlei: setal locations. Scale lines, 0.1 mm. P, M,, and I are prothorax, mesothorax, metathorax, and first abdominal segment. of non-nearctic species, would be necessary to know if 8-M and 7- setal acquisition is a constant character to be found in whole genera and possibly in the entire family. he criteria described above would be very useful to workers studying the population dynamics of mosquitoes. Acknowledgments his study was in part supported by postgraduate scholarships from the Natural Sciences and Engineering Research Council of Canada and the Direction G6n6rale de l'enseignement Sup6rieur du Qu6bec granted to R. Savignac. We thank C. Back and especially D. M. Wood, Biosystematics Research Institute, Ottawa, for their assistance in preparing the English manuscript and for their critical reviews.

5 Volume 113 HE CANADIAN ENOMOLOGIS Wyeomyia smithii M 2 0 P 300 3rd INSAR 17 4 M 2nd INSAR FIG. 3. Left lateral view of thorax of Wyeomyia smithii: setalocations. Scale lines, 0.I mm. P, M,, and I are prothorax, mesothorax, metathorax, and first abdominal segment. I able II. Numbers of setal support plates on each side of metathorax in five Canadian genera Support plates 2nd instar 3rd instar Aedes 1 2 Culex 1 2 Culiseta 1 2 Wyeomyia Anopheles

6 18 HE CANADIAN ENOMOLOGIS January1981 Aedes canadensis i i ' 2nd INSAR 4 o 9-12,30 FIo. 4. Left lateral view of thorax of Aedes canadensis: setal locations. Scale lines, 0.1 mm. P, M,, and I are prothorax, mesothorax, metathorax, and first abdominal segment. I References Belkin, J. N. and W. A. McDonahl A population of Uranotaenia anhydor from Death Valley, with descriptions of all stages and discussion of the complex (Diptera, Culicidae). Ann. ent. Soc. Am. 49: Bohart, R. M. and R. K. Washino Differentiation of second and third stage larvae of California Culex (Diptera: Culicidae). An. ent. Soc. Am. 50: Danks, H. V. and P.S. Corbet A key to all stages of Aedes nigripes and A. impiger (Diptera: Culicidae) with a description of first-instar larvae and pupae. Can. Ent. 105: De Oliveira, D. and M. Durand Head capsule growth in Culex territans Walker. Mosquito News 38: Dodge, H. R Studies on mos:luito larvae. I. Later instars of eastern North American species. Can. Ent. 95: Larval chaetotaxy and notes on the biology of oxorhynchites rutilus septentrionalis (Diptera: Culicidae). Ann. ent. Soc. Am. 57: Studies on mosquito larvae. II. he first-stage larvae of North American Culicidae and of world Anophelinae. Can. Ert. 98:

7 Volume 113 HE CANADIAN ENOMOLOGIS 19 Culex territans M 3rd INSAR p! z 6 s ø M 2nd INSAR I, 1 FiG. 5. Left lateral view of thorax of Culex territans: setal locations. Scale lines, 0.1 mm. P, M,, and I are prothorax, mesothorax, metathorax, and first abdominal segment. Eddieman, C. D Morphological and biometrical differentiation of the larval instars of mosquitoes. I. Culex territans. Ann. ent. Soc. Am. 60: Morphological and biometrical differentiation of the larval instars of mosquitoes. II. Orthopodomyia alba and O. signifera. Ann. ent. Soc. Am. 61: Harrison, B. A. and R. Rattanarithikul Comparative morphology of early larval instars of Aedes aegypti and A. seatoi in hailand. Mosquito Syst. 5: Hurlbut, H. S A study of the larval chaetotaxy of Anopheles walkeri heobald. Am. J. Hyg. 28: Knight, K. L Differentiation of the larval instars of Aedes sollicitans (Walker) and A. taeniorhynchus (Wiedemann)(Diptera: Culicidae). Proc. ent. Soc. Wash. 66: Knight, K. L. and J. L. Laffoon A mosquito taxonomic glossary. VIII. he larval chaetotaxy. Mosquito Syst. Newslett. 3: MacKenzie, D. W he thoracic chaetotaxy of the last three larval instars of four New England species of Aedes (Diptera: Culicidae). Ph.D. hesis, Univ. of Massachusetts. 59 pp. Makiya, K Head width of mosquito larvae for instar determination. Jap. J. san. Zool. 20: (In Japanese, English summary.)

8 - 20 HE CANADIAN ENOMOLOGIS January 1981 Cullseta melanura p "-'--- 3rd INSAR 1/ lo i i M 2nd INSAR I ' 6 6., I 6 o I I / FIG. 6. Left lateral view of thorax. of Culiseta rnelanura: setal locations. Scale lines, 0.1 mm. P, M,, and I are prothora::, mesothorax, metathorax, and first abdominal segment.! Reisen, W. K. and. F. Siddiqui Horizontal and vertical estimates of immature survivorship for Culex tritaeniorhynchus ([Diptera: Culicidae) in Pakistan. J. reed. Ent. 16: Smith, M. E. 1969a. he Aedes nosquitoes of New England (Diptera: Culicidae). II. Larvae: keys to instars, and to species exclusive of first instar. Can. Ent. 101: b. he Aedes mosqui:oes of New England. III. Saddle hair position in 2nd and 3rd instar larvae, with particular reference to instar recognition and species relationships. Mosquito Syst. Newslett. 1: Smith, S. M. and R. A. Brust Photoperiodicontrol of the maintenance and termination of larval diapause in Wyeomyia smithti (Coq.) (Diptera: Culicidae) with notes on oogenesis in the adult female. Can. J. Zool. 49: 10( Wood, D. M., P.. Dang, and 1. A. Ellis he insects and arachnids of Canada. Part 6. he mosquitoes of Canada (Dipterm Culicidae). Can. Dep. Agric. Publ pp. Yan, S.-L External structures of all larval instars of five common Culex species in aiwan. Chinese J. Microbiol. 10: (Received 20 March 1980)