Methods in Anopheles Research - MR4

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Chapter 3 : Specific Anopheles Techniques3.9 Family CulturePage 1 of 23.9 Family CultureMR4 StaffIntroductionFamily – or single family - culture is useful for many experimental plans or for the development of newstrains. It is particularly important when the mother’s genetics must be known, in genetic crossing, andwhen genotypic frequencies are of interest. If specific knowledge of the father’s genetics is important tostudies, pair matings must be used (Chapter 3.7.3) or inferred from progeny analysis. Otherwise, enmasse -mated females can be isolated for individual egging. It some cases, the genetics of the motherwill be determined based on the genotypes/phenotypes of her progeny.Another application of family culture is to establish wild colonies. In species complexes such asAn. gambiae, An. funestus and An. dirus where sympatric forms co-exist, it is essential to isolate thespecies of interest (Mpofu et al. 1993). By utilizing family rearing techniques, individual females areseparated so that eggs from a single pair are segregated, thus allowing the establishment of purebreedinglines that can be transformed into laboratory colonies.It is best to culture individual families in the dish in which the eggs were laid for the first couple of days toavoid moving fragile eggs/larvae. The Qorpak vial shown in Figure 3.9.1 are suitable for 100 or fewerprogeny, but larger numbers should be collected in larger cups to prevent early larval mortality. Thelarvae should be progressively transferred to larger containers and water volumes as they develop. It isnot uncommon to culture a family in three different containers before pupation to ensure good survival.A common error is to conclude that one’s family culture method is suitable even in the absence ofhatching data based on total egg hatch. Because L1s may die and decay rapidly, their presence can onlybe known based on a count of the total number of eggs that hatched. Particularly for frequency andsurvival data this is essential. Do not rely on counts of larvae even one day after hatching for quantitativedata.Obtaining eggs from reluctant femalesLow rates of oviposition often hinder successful family culture. Several methods have been developedthat have been shown to increase oviposition within the laboratory. The use of a dark oviposition dish ismore attractive than a clear or white dish in An. quadrimaculatus (Lund 1942), An. gambiae (Huang et al.2005), and An. arabiensis (MQB pers. comm.). In An. albimanus, wild caught blood fed females that wereallowed to oviposit in a 5 dram vial laid more eggs than those allowed to oviposit in a large cage (Baileyand Seawright 1984). The complete removal of one wing of a gravid female that is lightly anesthetized willpromote oviposition soon thereafter, though this is time consuming for the technician. This method is alast resort because mortality results.Rearing schedule for individual familiesIf you are starting with bloodfed material, begin with schedule at day 4.Day 1- Blood feed females.Day 2- No attention is required.Day 3- No attention is required.Day 4- Transfer gravid females to vials lined with filter paper (for example: Qorpack Bridgeville, PA. No.3891 containing strips of filter paper cut to size, Figure 3.9.2) and containing 1-2 cm of water.Day 5- Remove the females from their vials.Day 6- add 2 drops of a 2% w/v yeast slurry to each vial.
Chapter 3 : Specific Anopheles Techniques3.9 Family CulturePage 2 of 2Day 7-count hatch rate and transfer larvae to a larger container (Figure 3.9.2) containing 0.02% w/v finalconcentration of yeast (see Determining Egg Hatch Rates, Chapter 2).Day 8-observe, but feeding is usually not neededDay 9 through pupation-feed ground fish food or other larval diet. Monitor water quality carefully in thesesmaller pans as it is easier to accidentally over feed such a small number of larvae. Transfer to largercontainers as needed.Figure 3.9.1. This particular individual familyoviposition tube is modified from a chamberpurchased through Qorpack Bridgeville, PA.No. 3891.Figure 3.9.2. Examples of family rearing trays.ReferencesBailey DL, Seawright JA (1984) Improved techniques for mass rearing Anopheles albimanus. In: King EG(ed) Advances and challenges in insect rearing. USDA, New OrleansHuang J, Walker ED, Giroux PY, Vulule J, Miller JR (2005) Ovipositional site selection by Anophelesgambiae: influences of substrate moisture and texture. Med Vet Entomol 19:442-450Lund HO (1942) Studies on the choice of a medium for oviposition by Anopheles quadrimaculatus Say. JNatl Mal Soc 1:101-111Mpofu SM, Masendu HT, Kanyimo KH, Mtetwa C (1993) Laboratory colonization of Anophelesquadriannulatus from sympatry with other sibling species of the Anopheles gambiae complex inZimbabwe. Med Vet Entomol 7:122-126
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