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A Mosquito Systematics Vol. 14(3) A GUIDE TO THE GENERA OF MOSQUITOES (DIPTERA: CULICIDAE) OF THAILAND WITH ILLUSTRATED KEYS, BIOLOGICAL NOTES AND PRESERVATION AND MOUNTING TECHNIQUES by RAMPA RATTANARITHIKUL* CONTENTS Introduction Morphology Acknowledgments List of genera, subgenera in Thailand with their abbreviations and primary references (including keys) to that genus in Thailand or Southeast Asia Basic (common) immature habitats for mosquito genera and subgenera in Thailand.... Preservation and mounting techniques for mosquitoes... Morphology and abbreviations Adult morphology... Adult head.... Adult thorax and wing.... Male genitalia.... Pupal morphology.... Larval morphology... Illustrated keys to the genera of mosquitoes Adult... Male genitalia.... Pupa... Larva... Primary references *Department of Medical Entomology, US Army Medical Component, Armed Forces Research Institute of Medical Sciences (AFRIMS), APO San Francisco, CA 96346, or Rajvithi Road, Bangkok 4, Thailand. 140 INTRODUCTION During the last 20 years, the taxonomy and ecology of the mosquito fauna of Thailand has received much attention from professional taxonomists. During this period there have been many publications in scientific journals on the description, distribution, bionomics and medical significance of a single genus, subgenus, species group or a species. However, local public health workers and students concerned with the study and/or control of the mosquitoes in Thailand have often not had access to these publications and have been forced to work without keys, or at best, with outdated keys to the genera of mosquitoes in Thailand. In fact, there is a great need within Thailand for more basic information, particularly keys, for the mosquito fauna of Thailand. This publication has been prepared with the above needs in mind. The keys were prepared as simply as possible, using the most important characters. Hopefully, they will enable public health workers and students to quickly classify to genera, the adults, pupae, larvae and male genitalia of the mosquitoes of Thailand. In addition, references of particular importance for the identification of Thai mosquitoes have been included in Table 1. A total of 18 genera and 41 subgenera of mosquitoes are recognized from Thailand, and listed (Table 2) with their most common immature habitats. All scientific names used herein follow Knight and Stone (23) and Knight (18). Characters of both the adult and immature stages are very important for identification, in fact, some subgenera are best identified by characters of the immature stages. Accordingly, standard methods used at the AFRIMS for the preservation and mounting of immature and adult mosquito specimens have been included. Where possible, workers are encouraged to rear adults from the immature stages, preserving and mounting the 4th stage larval and/or the pupal exuviae as correlates of the adult. In this manner a specimen (adult, pupal and larval) can be identified by 3 keys or 4 if the adult is a male. If specimens that cannot be identified are encountered, the user is encouraged to submit these specimens to the Department of Medical Entomology, Armed Forces Research Institute of Medical Sciences (AFRIMS), Rajvithi Road, Bangkok. MORPHOLOGY The morphological characters used in the keys are illustrated for each couplet. Characters illustrated other than those referred to in the keys should not be used for identification. The terminology for the detailed characters of the female, male, male genitalia, pupa and larva follow Belkin (3), Reinert (44)) Harbach and Knight (lo), Knight (17) and Knight and Laffoon (20, 21, 22). Since the morphological terminology for mosquitoes is extensive and may be confusing, some of the most commonly used morphological terms are A Mosquito Systematics Vol. 14(3) listed below, and where possible, (IO). defined according to Harbach and Knight AEDEAGUS - In most male mosquitoes, the central body of the phallosome serving as the intromittent organ. ALVEOLUS - A cup-like depression (socket) bearing a seta or scale. BASAL MESAL LOBE (BML) - In male mosquitoes, a small lobe or sclerite located basomesally on the gonocoxite; usually connected ventrally with its mate; possibly homologous with the claspette. (See PARABASAL LOBE). CAUDAD - Posterior part of the body, away from the head. CEPHALAD - Anterior part of the body, toward the head. CHAETOTAXY - The study of the arrangement, branching and numbering of the setae on the exoskeleton of insects. CUTICULAR PROJECTION - Any elongate process jutting from the outer surface of the cuticula. There are two basic types of cuticular projections: (I) SETAE, which are articulated processes arising from a basal alveolus, and (2) SPICULES, which are non-articulated, continuous processes of the cuticula. EMARGINATE - Having a notched margin or tip. FILAMENT - A long slender, flexible spicule which gradually tapers to a point or is of equal diameter throughout. FRINGE - A border projecting from a margin, usually of fairly even length and composed of setae, spicules or scales. MAXILLARY PALPUS - The teleopodite of the maxilla. In adult mosquitoes, inserted immediately below the clypeus and laterad to the base of the proboscis; varies widely in form according to genus and sex: primitively consisting of five palpomeres. MESAD - Toward the midline of a bilaterally symmetrical body. MICROTRICHIUM - One of the minute, slender, tapered, flexible spicules closely covering the wing membrane; characteristically bent so that they extend more or less parallel to the longitudinal veins with their apices pointed toward the wing margin. OPISTHOPHALLAS (QP) - A transverse lobiform element of the phallosome located between the phallus and proctiger. PARABASAL LOBE (PBL) - In male Anophelinae, a variably developed lobe located dorsobasally (prerotation sense) on the gonocoxite; bearing one or more strongly developed parabasal setae. PHALLOSOME (PH) - In male mosquitoes, the complex structures surrounding the gonopore between the proctiger, gonocoxites and sternum IX; presumably consisting primitively of the basal pieces, parameres, prosophallus, phallus and opisthophallus, e. g., in Aedes subgenus VerraZZina; usually comprised of the basal pieces, parameres and aedeagus, but including the lateral plates in Culex; the homologies of the various components are unknown. PHALLUS (Ph) - The median lobe of phallosome located between the prosophallus and opisthophallus. 142 POSTTARSUS - The terminal segment of the arthopod leg distal to the tarsus. PROCTIGER (Pr) - In insects with poorly defined segmentation behind the genital segments, the entire complex formed by the telson and the postgenital segments (abdominal segments X and XI). In such insects, the telson and abdominal segment XI are entirely membranous. PROSOPHALLUS (PO) - A ventral lobelike element of the phallosome located between the bases of the gonocoxites. PULVILLUS - One of two padlike or divided lobes on the posttarsus of some insects; one arising below the base of each unguis. SCALE - A modified seta comprised of a slender basal stalk, pedicel, and an expanded and/or flattened distal part, squame; all scales have longitudinal ridges and arise from alveoli which are usually minute. Two main types of scales are recognized in mosquitoes by cross section of the squame. If the squame is round or elliptical, the scale is piliform; if it is thin and flat, the scale is lamellar. Many forms of lamellar scales exist but only one type of piliform scale. Asymmetrical - A form of lamellar scale which is obviously unevenly developed on opposite sides of a plane which is parallel to the plane of the squame. SERRATION - A recurved denticle-like process; a series of such processes is termed serrations. SETA - A cuticular projection which arises from a basal alveolus. Aciculate - Furnished with slender needlelike processes along the stem; the processes are somewhat flexible but often appear to be rigid. Barbed - With minute, short, heavy, pointed processes projecting obliquely from the surface. Bifid - Divided into two parts; forked. Pahzate - With flattened, movable, usually horizontal branches. SteZZate - Tufted; with numerous stiff branches projecting at various angles from a single base; branches may be simple, barbed or aciculate. SPICULE - A non-articulated cuticular projection, directly continuous with the cuticula. SPINE - A very large, sturdy, immovable spicule, with a sharp or narrowly rounded tip. TRAGUS - A more or less elaborate lobe sometimes occurring on the rim of the pinna of a laticorn trumpet. UNGUIS - An anterior or posterior claw of the arthropod posttarsus; either or both may be absent; usually both are present on the posttarsus of mosquitoes; referred to as fore-, mid- or hindungues as appropriate. Abbreviations are used in the key when referring to the pupal and larval chaetotaxy of head, thorax and abdomen. Examples are as follows: 1-A = Seta 1 on the larval antenna 2-c = Seta 2 on the larval head 7-P = Seta 7 on the larval prothorax A Mosquito Systematics Vol. 14(3) s = Seta 1 on the larval siphon 4-x = Seta 4 on the larval abdominal segment X l-pa z Seta 1 on the pupal paddle ACKNOWLEDGMENTS 4 I wish to express my deep appreciation to Dr. Douglas J. Gould, former Chief, Department of Medical Entomology, US Army Medical Component, SEATO, Bangkok, for suggesting the need for this study. I am much indebted to LTC Bruce A. Harrison, former Chief, Department of Medical Entomology, Armed Forces Research Institute of Medical Sciences (AFRIMS), and current Chief, Walter Reed Biosystematics Unit, Smithsonian Institution, for many helpful suggestions and consultations throughout the study, his great assistance in the initial reviews and in arranging for the final critical review, and for overseeing the final preparation for publication. I extend special thanks to Mr. E. L. Peyton, formerly assigned to the Department of Medical Entomology, US Army Medical Component, SEATO, Bangkok, now Research Entomologist, Medical Entomology Proj ect, Smithsonian Institution, for reviewing the manuscript. Special thanks are also due Mr. Kol Mongkolpanya and Mr. Prajim Boonyakanist for providing detailed information on the mosquito habitats in Thailand. I also appreciate the assistance of the field teams and laboratory assistants for collecting, rearing, and preparing specimens for this study; Mr. Chumnong Noigamol, Mr. Ruan Thaopha, Mr. Sanit Nakngen, Mrs. Prasertsri Rohitaratana and Mrs. Suda Ratanawong. Finally, special thanks are due Mrs. Prachong Panthusiri for preparing the illustrations and to Mrs. Nipaporn Nimsombun and Mrs. Khanitha Rojanasthien for typing the manuscript. NOTE After the manuscript was submitted the following revision of Paraedes was published. This publication should serve as the primary reference for this subgenus. Reinert, 3. F Med. Ent. Studies - XVI. A revision of the subgenus Paraedes of the genus Aedes (Diptera: Culicidae). Contrib. Amer. Entomol. Inst. (Ann Arbor). Vol. 18(4):1-91. 144 Table 1. List of genera, subgenera in Thailand (18,23) with their abbreviations (45) and primary references (including keys) to that genus in Thailand or Southeast Asia. c - - I GENERA SUBGENERA PRIMARY REFERENCES _~_-._-~-V Anopheles - An Anopheles - An0 5, 12, 30, 35, 36, 37, 50 Cellia - Cel 5, 11, 34, 35, 36, 37, Aedeomyia - Ad Aedeomyia - Ady 2, 30, 56, Aedes - Ae Aedimorphus - Adm 2, 14, 30, 40 Ayurakitia - Ayu 39, 56 Bothaella - Bot 43 Cancraedes - Can 2, 26 Christophersiomyia - Chr 1, 2, 27 Diceronyia - Die 2, 27, 38, 42 Edwardsaedes - Edw 46 Finlaya - Fin 2, 6, 16, 19 Isoaedes - Isa 49 Lorrainea - Lor 27 Mucidus - Muc 2, 28, 58 Neomelaniconion - Neo 2, 28 Ochlerotatus - Och 2, 28 Paraedes - Par 26, 48 (See note, p. 143) Rhinoskusea - Rhi 2, 26, 47 Stegomyia - stg 2, 13, 14, 15 Verrallina - Ver 2, 7, 8, Armigeres - Ar Armigeres - Arm 2, 30, 56 Leicesteria - Lei 2, 24, 56 Mosquito Systematics Vol. 14(3) _-_ ~_II--- GENERA =I-- SUBGENERA PRIMARY REFERENCES -...l Heizmannia - Hz Heizmannia - Hez 2, 29, 30, 41 Mattinglyi - Mat 29, Udaya - Ud 30, Culex - cx Culex - cux 2, 4, 30, 53 Culiciomyia - Cui 4, 52, 55 Eumelanomyia - Eum 51 Lophoceraomyia - LoP 54 Lutzia - Lut 4 Thaiomyia - Tha 4 8. Ficalbia - Fi 2, 25, Mimomyia - Mi Etorleptiomyia - Eto 2, 25, 30 Mimomyia - - Mim 2, 25 Ingramia - Ing 25, Hodgesia - Ho 2, 30, Coquillettidia - Cq Coquillettidia - coq 2, 30, Mansonia - Ma Mansonioides - Man 2, 30, Orthopodomyia - Or 2, 30, Malaya - Ml 2, 30, Topomyia - To Topomyia - Top 2, 30, 56 Suaymyia - Sua Tripteroides - TP Tripteroides - Trp 2, 9, 30, 31, Uranotaenia - Ur Pseudoficalbia - Pfc 2, 30, 32, 33 Uranotaenia - Ura 2, Toxorhynchites - TX Toxorhynchites - Tox 2, 30, 56 148 PRESERVATION AND MOUNTING TECHNIQUES FOR MOSQUITOES 1. WHOLE LARVAE 1. Kill larvae in hot water (not boiling) remove promptly. 2. Store in small vial containing 75-80% ethanol (ethyl alcohol). 3. Transfer specimens from alcohol to cellosolve for 15 minutes or more (dark specimens can be stored in cellosolve for 8 hours or overnight). 4. Lift the specimen from cellosolve* and place on the center of glass microscope slide with the dorsal side up. 5. Drop a small amount of Canada balsam** on specimen. Mount specimen dorsal side up with the head pointing down, arrange head, thorax and abdomen in natural position, then cut the abdomen between segment VI and VII (using surgical blade No. 25). Place the terminal segments with siphon to the left in culicine larvae or segment X to the right in anopheline larvae. (See Plate I). _ 6. Add more Canada balsam on specimen and check the arrangement of setae and larval position, then carefully cover specimen with a 22 mm rectangular cover glass. 7. Dry in drying oven at 45o-550C for 2 weeks or more. 2. WHOLE PUPAE 1. Follow steps l-5 described for mounting whole larvae. 2. Separate cephalothorax from the metanotum and abdomen. Mount specimen pointing down, place metanotum and abdomen dorsal side up then turn cephalothorax left side up and place below the metanotum. (See Plate I). 3. Add more Canada balsam on specimen, check specimen for correct position. Cover specimen with a 15 mm round cover glass. 4. Dry in drying oven at OC for 2 weeks or more. 3. LARVAL AND PUPAL EXUVIAE The 4th larval and pupal exuviae from an individually reared adult should be mounted on the same slide. 1. Store in 75-80s alcohol. 2. Transfer specimens into cellosolve for 15 minutes. 3. Lift the specimen from cellosolve placing it on a glass microscope slide, the larval exuvia on the left and pupal exuvia on the right (pointing head down and dorsal side up). 4. Drop a small amount of Canada balsam on specimens. Arrange and spread body and setae of larval exuvia into better position, then separate the *Dilute cellosolve may turn white when adding Canada balsam on specimen. To avoid this problem, use facial tissue to blot off the excess cellosolve from the specimen before adding Canada balsam on specimen. (Touch specimen carefully and softly with small piece of facial tissue). **Euparal can be used instead of Canada balsam. Thick Canada balsam can be thinned down with xylene and Euparal with Euparal essence. Mosquito Systematics Vol. 14(3) pupal cephalothorax just cephalad of wing sheath, leave metanotum attached to abdomen. Open the cephalothorax and mount the ventral side up, place below metanotum. (See Plate I). 5. Add more Canada balsam, check position of larval and pupal exuviae then cover specimens with a 15 mm round cover glass. 6. Dry in drying oven at 45o-55OC for 2 weeks or more. 4. ADULTS 1. After emergence, adults should be held for at least 24 hours before killing. Dried specimens should be relaxed in a relaxing jar for at least 2 hours so that the appendages will not break when handled. 2. Kill in chloroform* killing bottle. (Ethyl acetate keeps specimens relaxed longer). 3. Using a small amount of Ambroid cement** on tip of paper point, glue specimen on the right side of thorax with legs toward pin, wing and body should not be attached with paper point and glue, and point should not project beyond scutum. (See label, Plate I). 4. Pinned specimens should be kept in Schmitt boxes. Paradichlorobenzene* or napthalene* is needed for each box to protect specimens from being eaten by beetles, cockroaches, mites and other insects. The former has both repellent and insecticidal properties but does not last as long as the latter which basically acts as a repellent. 5. GENITALIA 1. Place adult in relaxing jar for 3-5 hours*** (Can use a jar with boiling-steaming water for minutes). 2. Clip abdomen between 6th and 7th segments using a fine dissecting scissors. 3. Place the genitalia in a small screw cap vial containing 10% KOH**** and a small drop of 40% detergent to sink the genitalia in the KOH. 4. Put the vial in a beaker of boiling water (but do not boil contents in vial), or leave the vial in drying oven at 45o-550C for 1 hour. 5. Transfer the genitalia to cellosolve for IO-15 minutes. 6. Place the specimen on the center of a glass microscope slide. Dissection of the genitalia should be made in cellosolve. Dissect the genitalia from the 7th and 8th abdominal segments and place below the segments, tergal side up with the gonostylus pointing down. (See Plate I). 7. Position each piece of the genitalia in a minute drop of Canada balsam. Dry slide in oven overnight. *CAUTION! Chloroform and Ethyl Acetate are toxic and dangerous to breathe. These chemicals are stored in liver tissues and may cause health problems if used frequently. Always use in well ventilated areas. ** Ambroid cement should be thinned down with amyl acetate. ***Specimens are normally mounted on points or pinned before this procedure.. ****Potassium hydroxide. Add more Canada basam over specimen and place 2-3 tiny pieces of broken cover glass surrounding the specimen and cover with a 12 mm round cover glass. 9. Dry in drying oven at 450,550C for 2 weeks or more. 6. STAINING GENITALIA I. Follow genitalia mounting techniques steps Remove and place genitalia in 5% acetic acid and 1-2 drops of acid fuchsin solution* overnight or leave in the oven for another hour. 3. Remove and place in cellosolve (can be stored in this). 4. Follow genitalia mounting techniques. 7. LABELING (Plate I) 1. Label the slide with 2 labels: a. Left lube1 contains: 1. Country, collection number 2. Province, date collected 3. Location 4. Collector s name 5. Habitat 6. Slide number b. Right lube1 contains: I Label the adult size or smaller. a. UPPer b. Lower Genus Subgenus Species Person making determination with 2 labels. Each label should be l/4, x l/2 in label contains: Country, province Location Collection Number Date/Year. label contains: Genus, subgenus and species Sex Person making determination *Acid fuchsin solution: acid fuchsin.05 gm. 10% hydrochloric acid - 25 cc. distilled water cc. Mosquito Systematics Vol. 14(3) THAILAND: Chumphon. 6 Sop71 Bon Kon Thor Coil: Kol HBT: water jar Slide # F-8204 / Ip( w Det. (Stegomyio) ae9ypti RR LARVA PUPA THAlLAND:07819-I Chiong Moi. 4 Jul78 Doi lnthonon Coil: Harrison HBT: bonano oxil Slid. # F-7084 Det. RR Malay0 jacobsoni THAILAND: Chiang Mai. Doi lnthonon Coil: Harrison Gonitolia Jul78 Malaya jocobsoni Slide # 2481 Det. RR J I LARVAL AND PUPAL EXUVIAE GENITALIA -- k- 0 -a :_ -- I THAILAND: Huai Kuum Chon Buri COII &.(Cel.)* 9 - Det. RR ADULT PLATE I 152 ADULT MORPHOLOGY FLAGELLOMERE ANTENNA MAXILLARY (FLAGELLUM) PALPUS SCUTELLUM MESOPOSTNOTUM CEkCUS // PLATE I( HIND III LEG Mosquito Systematics Vol. 14(3) HEAD CULICINAE ANOPHELINAE erect scales on occiput & erect scales on vertex Jlf broad decumben VERTEX fronto scales on vertex arrow decumbent tales on vertex. setoe MAXILLARY PALPUS PROBOSCIS ANTERIOR ASPECT LATERAL ASPECT PLATE III 154 THORAX antepronotum anterior promontory fossol area anterior dorsocentro posterior fossol scutum mesomeron coxo m SCUTELLUM MESOPOS
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