Methods in Anopheles Research Fourth Edition 2014 Table of Contents Preface Chapter 1 : Insectary Operation 1.1 Equipping and Operating an Insectary 1.2 Cleanliness and General Maintenance 1.3 Scheduling and Regulating Your Work Load 1.4 Maintaining Stock Purity 1.5 Insectary Manager Responsibilities 1.6 Trapping in the Insectary Chapter 2 : Anopheles Laboratory Biology and Culture 2.1 Behavior and Physiology of Anophelines in the Laboratory 2.2 Infections in Mosquito Cultures 2.2.1 PCR to Detect Microsporidian Infections in Laboratory Colonies 2.2.2 PCR to Detect Fungal Infections in Laboratory Colonies 2.3 Modifying Fecundity, Longevity and Size 2.4 Anopheles Culture 2.4.1 Collecting Anopheles Eggs 2.4.2 Bleaching Anopheles Eggs 2.4.3 Hatching Anopheles Eggs 2.4.4 Determining Egg Hatch Rates 2.4.5 Estimating the Number of Eggs and Larvae 2.4.6 Anopheles Larval Culture 2.4.6.1 Standardized Larval Culture 2.4.7 Separating Larvae and Pupae 2.4.8 Anopheles Adult Caging 2.4.9 Anopheles Adult Diet 2.4.10 Blood Feeding : Membrane Apparatuses and Animals 2.5 Basic Anopheles Mendelian Genetics 2.6 Basic Anopheles Population Genetics Chapter 3 : Specific Anopheles Techniques 3.1 Embryonic Techniques 3.1.1 Microinjection Methods for Anopheles Embryos 3.1.2 Anopheles Embryo Fixation 3.1.3 Establishing Cell Lines from Anopheles spp. Embryonic Tissues 3.2 Eye Color Mutant Screening 3.2 Pupal/Larval Sexing Table of Contents 3.4 Mosquito Anesthesia 3.5 Protocol for 96 Well gDNA Extraction 3.6 Determination of Lipid, Glycogen, and Sugars in Mosquitoes 3.7 Anopheles Mating 3.7.1 Mating : General Considerations 3.7.2 Forced Copulation 3.7.3 Pair Matings 3.7.4 An. gambiae and arabiensis Mating Status Determination 3.8 Forced Oviposition for Anopheline Mosquitoes 3.9 Family Culture 3.10 Picogreen Quantification of DNA from a Single Mosquito 3.11 Microinjection of Adult Anopheles gambiae 3.12 Hemocyte Collection Protocol for Anopheles Chapter 4 : Stock Authentication 4.1 Authentication by Morphological Characteristics 4.2 Authentication by PCR 4.2.1 Anopheles gambiae TEP 1 Assay 4.2.2 Anopheles arabiensis ND5 Molecular Authentication 4.2.3 Anopheles gambiae White gene 4.2.4 Anopheles gambiae APL1A Assay Chapter 5 : Insecticide Resistance Monitoring 5.1 Insecticide Resistance Bioassays 5.1.1 Larval Insecticide Resistance Assays 5.1.2 WHO Paper Testing 5.1.3 CDC Bottle Bioassays 5.1.4 WHO Cone Assay 5.2 Microplate Enzyme Activity Assays Introduction 5.2.1 Microplate Insensitive Acetylcholinesterase Assay 5.2.2 Microplate Glutathione s-transferase Assay 5.2.3 Microplate Nonspecific Esterase Assay 5.2.4 Microplate Oxidase Assay 5.3 Insecticide Resistance Allele Assay by PCR 5.3.1 Knockdown Resistance - Anopheles gambiae 5.3.2 Knockdown Resistance in An. gambiae (MR4 2012) 5.3.3 Dieldrin Resistance - An. gambiae and An. arabiensis 5.3.4 ACE-1 Resistance in An. gambiae Table of Contents 5.3.5 ss-PCR Assay to Detect ACE-1 in An. gambiae 5.4 Mechanical Aspirator for Transferring Insects used in Bioassays Chapter 6 : Dissection Techniques 6.1 General Dissection Buffers 6.2 Rapid Larval Midgut Extraction 6.3 Larval Midgut Vivisection 6.4 Adult Male Testes Dissection 6.5 Dissecting Plasmodium-infected Mosquitoes 6.5.1 Midgut 6.5.2 Salivary Glands 6.6 Examination of Ovaries by Tracheal Distension to Determine Parity 6.7 Dissecting Spermathecae to Determine Insemination Status 6.8 A. gambiae s.l. Ovarian Polytene Chromosome Preparation 6.9 A. gambiae s.l. Salivary Gland Chromosome Preparation Chapter 7 : Taxonomy and Systematics 7.1 Taxonomic Mounting Techniques 7.2 Ribosomal DNA PCR Assays 7.2.1 ITS2 PCR 7.2.2 28S Subunits PCR 7.2.3 General PCR for the 18S subunit in anophelines 7.3 Mitochondrial DNA PCR Assays 7.3.1 COI/COII PCR 7.3.2 Cytochrome B PCR Chapter 8 : Field Techniques 8.1 Plasmodium Detection by PCR in Mosquitoes 8.1.1 Single Step Molecular Identification of Plasmodium in Anophelines 8.2 Sporozoite ELISA 8.3 Molecular Identification of Bloodmeals in Mosquitoes 8.4 Species Complex Identification by PCR 8.4.1 Anopheles gambiae complex (Scott et al.) 8.4.2 Anopheles gambiae complex (Fanello et al.) 8.4.3 Combined An. gambiae species ID and rDNA type (Wilkins et al.) 8.4.4 Anopheles funestus complex (Koekemoer et al.) 8.4.5 Anopheles funestus complex (Wilkins et al.) 8.4.6 Anopheles minimus complex Table of Contents 8.4.7 Anopheles quadrimaculatus complex (Cornel et al.) 8.4.8 Anopheles quadrimaculatus complex (Levine et al.) 8.4.9 SINE based assay for An. gambiae M and S forms (Santolamazza et al.) 8.4.10 Identification of An. gambiae and An. arabiensis from degraded samples 8.5 RT-PCR Assays 8.5.1 Vector Population Monitoring Tool using Real Time PCR 8.5.1.1 An. gambiae s.l. species complex ID assay 8.5.1.2 Plasmodium detection assay 8.5.1.3 Knock down resistance (kdr) assays 8.5.1.4 Insensitive acetylcholinesterase (iAChE) assay 8.5.1.5 Resistance to dieldrin (rdl) assay 8.6 Molecular Karyotyping PCR Assays 8.6.1 Anopheles gambiae 2La Inversion (White et al.) 8.6.2 Anopheles gambiae 2Rb Inversion (Lobo et al.) 8.6.3 Anopheles merus 2Ro Inversion (MR4) 8.7 LAMP Assay for Anopheles gambiae s.l. Chapter 9 : Guidance for Obtaining Living Materials 9.1 Permits and Regulations Chapter 10 : The Care and Maintenance of Aedes Laboratory Colonies 10.1 Aedes Culture 10.1.1 PCR to differentiate between various aedine species 10.1.2 Mitochondrial PCR Assays for Aedes Mosquitoes Chapter 11 : The Care and Maintenance of Culex Laboratory Colonies 11.1 Culex Culture 11.1.1 PCR to differentiate between members of the Culex pipiens complex (Fonseca et al.) 11.1.2 PCR to detect Wolbachia endosymbionts in mosquitoes 0-1 Preface.doc Page 1 of 2 Preface This training manual represents the latest incarnation of materials I began developing years ago for the instruction of personnel working with me who were given the dicey proposition of culturing Anopheles mosquitoes. Originally, it was entitled “Raising Mosquitoes for Fun and Profit,” and then it became “Anopheles Mosquitoes for Insectary Personnel: An Introduction to Cleanliness, Biology, Culture, Genetics, and Productivity” which had the nifty graphic that you see here. As is perhaps inevitable, the latest incarnation doesn’t have the same fun name as the original, but we have broadened the contributing authors list and refined the content into something that we hope will better serve the needs of a larger audience. We also hope it will provide the basis for a manual that is continually growing in content and quality. Therefore, the complete manual has no version or edition number. New sections or revisions may appear on the web at any time. We acknowledge the contributions of many who have cultured mosquitoes and published their experiences such as Trembley (1944) and Gerberg (1994). We are extremely indebted to the continuously useful two edition set of books by Clements on mosquito biology from which we have unashamedly extracted extensive information (1992; 1999). We highly recommend their purchase by any laboratory culturing and studying mosquitoes. This inaugural version was stimulated by the needs of the first of many (we hope) courses entitled “Advanced Techniques in Anopheles Culture” conducted at the Centers for Disease Control and Prevention in Atlanta, GA USA in 2007. No doubt its quality will be improved by comments and response from the attendees. Previously, it was shaped by many people who listened to my sermons, forgave my pride (in mosquito culture!?), tolerated my boring lectures, and commented on ways to improve the course including the following: Safia Ali Sarah Jordan Wolfgang Schmeid Hervé Bossin Bart Knols Osama Seidahmed Gena Lawrence Dwight Mount Sharon Soliban Genelle Grossman Matt Murphy Theresa Stevens Andrew Hammond Doug Nace Janis Thailayil Michelle Helinski Pamela 'P2' Patterson Julian Turk Rebecca Hood Cristina Rafferty Tyrone Williams Without Liz Wilkins and Paul Howell, this content would not be possible. They worked to collect techniques, test them, and edited the sections to a uniform style. They are super people to work with and I cannot say enough good about their efforts on this and in the insectary where they have demonstrated skill applying the principles described here. Where you see authors as ‘MR4 staff,’ that’s Paul, Liz, and me. We invite researchers to contribute chapters to supplement what is presented here and to offer corrections and refinements. This manual is unashamedly full of prejudice and biases due to certain experiences. These very qualities will make it personal and enjoyable. 0-1 Preface.doc Page 2 of 2 May it serve well those working to reduce malaria and other vector-borne diseases by the study of their hosts. May, 2007 Mark Q. Benedict CDC, Atlanta USA References Clements AN (1992) The Biology of Mosquitoes: Development, Nutrition and Reproduction. Chapman & Hall, London Clements AN (1999) The Biology of Mosquitoes: Sensory Reception and Behaviour. CABI Publishing, New York Gerberg EJ, Barnard DR, Ward RA (1994) Manual for mosquito rearing and experimental techniques, revised ed. American Mosquito Control Association, Inc., Lake Charles Trembley HL (1944) Mosquito culture technique. Mosquito News 4:103-119 Preface to Second Edition When Mark Benedict and the MR4 Vector Activity Team launched the first edition of Methods in Anopheles Research in 2007, they never imagined its enormous success. This manual has become one of the most accessed items in the MR4 Vector Activity website, providing practical techniques to the intrepid researcher tasked with the “dicey” activity of rearing and investigating anopheline mosquitoes.