Quantifying Fitness Costs in Transgenic Aedes aegypti Mosquitoes

Summary

The present protocol describes how to measure common life parameter data in Aedes aegypti mosquitoes, including fecundity, wing size, fertility, sex ratio, viability, development times, male contribution, and adult longevity. These measurements can be used to assess the fitness of transgenic mosquitoes.

Abstract

Transgenic mosquitoes often display fitness costs compared to their wild-type counterparts. In this regard, fitness cost studies involve collecting life parameter data from genetically modified mosquitoes and comparing them to mosquitoes lacking transgenes from the same genetic background. This manuscript illustrates how to measure common life history traits in the mosquito Aedes aegypti, including fecundity, wing size and shape, fertility, sex ratio, viability, development times, male contribution, and adult longevity. These parameters were chosen because they reflect reproductive success, are simple to measure, and are commonly reported in the literature. The representative results quantify fitness costs associated with either a gene knock-out or a single insertion of a gene drive element. Standardizing how life parameter data are collected is important because such data may be used to compare the health of transgenic mosquitoes generated across studies or to model the transgene fixation rate in a simulated wild-type mosquito population. Although this protocol is specific for transgenic Aedes aegypti, the protocol may also be used for other mosquito species or other experimental treatment conditions, with the caveat that certain biological contexts may require special adaptations.

Introduction

Survival of the fittest is the Darwinian idea that individuals who harbor genes best adapted to their environment will pass those genes to subsequent generations¹. This means that it is fitness that determines whether their genes will survive. This more than 150-year-old concept is perhaps the most significant determinant of engineering a successful gene drive in transgenic mosquitoes. Gene drives, or the super-Mendelian inheritance pattern of a selfish genetic element that allows it to spread through populations², are being explored for genetic pest management³. In the context of vector control, this strategy aims to either replace wild-type (WT) arthropods with those resistant to pathogens (population modification) or eliminate them all together (population suppression)⁴. However, transgenic mosquitoes often exhibit fitness costs (also called genetic load) in comparison to their WT counterparts, which means that the transgene will be lost in populations that can outcompete them. Coupling transgenes with a gene drive system is therefore necessary to offset any fitness costs and push the transgene through the population at levels greater than expected from typical Mendelian inheritance⁴.

Laboratory studies across mosquito vector species have shown that transgenes often display fitness costs^5,6,7. For example, Irvin et al. measured various life parameters in Aedes (Ae.) aegypti engineered to express enhanced green fluorescent protein (eGFP) or transposase genes under Drosophila actin 5C or synthetic 3XP3 promoters, and compared them to the wild-type Orlando strain (the same genetic background from which they were derived)⁵. Most notably, they found that all transgenic strains had significantly reduced reproductive fitness⁵. Dependent on the transgene, some Anopheles (An.) mosquitoes expressing transgenes that inhibited Plasmodium parasite development also exhibited fitness costs⁶. Specifically, An. stephensi expressing a bee venom phospholipase (PLA2) under constitutive or bloodmeal-inducible promoters laid significantly fewer eggs compared to controls⁶. Those authors also found that transgenic Anopheles expressing a different transgene, an SM1 dodecapeptide tetramer did not exhibit fitness costs, leading them to conclude that transgene-conferred fitness costs are dependent on, at least likely in part, the effect of the transgenic protein produced⁶. Indeed, fitness costs may be attributed to transgene products, positional effects, off-target effects, insertional mutagenesis, or inbreeding effects in laboratory-reared strains⁷. Gene drives must therefore be robust enough to offset these transgene-induced fitness costs while also avoiding the development of insertions and deletions (indels) that block the drive itself.

Developmental or reproductive fitness costs can be measured in laboratory or cage studies⁷, with a caveat being that unknown factors in the field may also have an impact. Nonetheless, controlled fitness studies are an important first step when planning or evaluating genetically modified mosquito releases, such as a gene drive program, to determine whether the transgenic line will persist over subsequent generations. For example, Hammond et al. evaluated An gambiae CRISPR/Cas9-based-gene drives meant to disrupt the genes necessary for female fertility⁸. Through cage studies maintained for at least 25 generations, the authors found that the mosquitoes incurred gene-drive resistant alleles that blocked CRISPR-targeted cleavage and restored female fertility⁸. Their modeling efforts suggested that fertility in females heterozygous for the gene drive had the most dramatic impacts on gene drive fixation in simulated conditions⁸. Along these same lines, Ae. aegypti (Higgs' White Eye strain, HWE) engineered to express autonomous gene drive cassettes under different promoters or at different intergenic loci exhibited different rates of gene drive fixation in simulated populations⁹. Using MGDrivE modeling¹⁰, combined with measured rates of indel formation, maternal effects, and laboratory-collected life parameter data, the authors found that mosquito fitness most strongly influenced the persistence of the gene drive in simulated conditions⁹. Fitness costs that are most likely to impair gene drive efficiency are attributable to somatic Cas9 (over) expression or from the gene that is targeted, particularly in heterozygotes, rather than the intrinsic drive itself^{11,12,13,14,15,16,17}.

Given its significance, fitness is an important factor for the ability of a transgenic line to persist over subsequent generations, and it can be used as an indicator for any physiological effects associated with a transgene. For example, off-target effects may be associated with fitness costs. In this case, backcrossing a transgenic line for several generations is recommended. Furthermore, crossing the transgenic line with one that is reflective of a field population may also be necessary to investigate how well the transgenic population can compete in the real world. To quantify fitness costs so that they can be comparable, this manuscript provides simple protocols for measuring common life history traits in Ae. aegypti mosquitoes, with a particular emphasis on fitness costs associated with transgenes, so that such studies can be more easily reproduced. Protocols include fecundity, fertility, sex ratio, viability, development times, male contribution, and adult longevity. Measuring wing length and area was also chosen as a fitness measurement as it correlates with thorax length^18,19 and body size measurements, which is directly linked to bloodmeal size, fecundity, and immunity^20,21. Although there are many ways to assess fitness, these parameters were chosen because they reflect reproductive success, are simple to measure, and are commonly reported in the literature.

Protocol

NOTE: This protocol is written for transgenic and wild-type Ae. aegypti lines that have been previously validated and established. For more information on generating transgenic Ae. aegypti, see Kistler et al.22 and Coates et al.23. All experiments outlined below were performed under standard Ae. aegypti rearing procedures. Mosquitoes were maintained at 28 °C with 75%-80% relative humidity and a 12 h …

Representative Results

Following the above protocol, the fitness of two mosquito lines were evaluated: (1) CRISPR/Cas9-mediated knock out of the Ae. aegypti D7L1 (AAEL006424) salivary protein and (2) Ae. aegypti lines expressing autonomous CRISPR/Cas9-mediated gene drives9. In the case of the former, a homozygous D7L1 knock-out line was established by exploiting the non-homologous end-joining pathway (NHEJ) to generate the disruption after microinjecting embryos with sgRNAs specific to the D7L1</em…

Discussion

Ae. aegypti fitness studies are often performed in the laboratory to assess fitness costs associated with transgenic cargo (e.g., gene drive elements) or gene knock outs, as discussed in this manuscript; however, these studies may be performed for a variety of purposes—any that aim to evaluate the health of an Ae. aegypti group, such as Wolbachia-infected^30,31, insecticide resistant^32,</s…

Materials

1 oz. translucent plastic souffle cups	WebstaurantStore	301100PC
2 oz. translucent polystyrene souffle cups	WebstaurantStore	760P200N
3 mL plastic pipettors	Cornin	357524
50 mL conical tubes	Any brand
64 oz. white double poly-coated paper food cup	WebstaurantStore	999SOUP64WB	for mosquito enclosement
65 mm lens	Canon	MP-E 65mm f/2.8 1-5x Macro Photo	Canon Macro Photo MP-E 65mm, 7D-65mm-1X; zoom=1, 200, 6.3, ISO=100; for photographing wings or egg papers, although other cameras are likely sufficient
Aedes aegypti mosquitoes	BEI	multiple strains as eggs are available
Artifical membrane feeders	https://lillieglassblowers.com/	Meduim membrane feeder, Custom made, 33mm	Chemglass also offers, but sizes are wrong for us. Ours are about 3 cm?
ATP	MP Biomedical	ICN15026605	Any good quality ATP, 10mM filter sterile aliquots at -20
Autoclave			for sterilizing water for hatching
Canon EOS 7D camera	Canon	3814B004	for photographing wings or egg papers, although other cameras are likely sufficient
defibrinated sheep blood	Colorado Serum Co.	60 ml, every 2 weeks	https://colorado-serum-com.3dcartstores.com/sheep-defibrinated
Dual Gooseneck Microscope Illuminator	Dolan Jenner Fiber-Lite 180	181-1 System
Ethanol
Forceps	Dumont	5SF
Gauze		omnisorb ii	4" non-woven sponges
glass microscope slide	Fisher Scientific	12-544-2
Glass Petri dishes, 100 × 15 mm	VWR	75845-546	for anesthesizing/manipulating mosquitoes on ice
Hogs' gut	Any Deli		we buy in bulk, split, wash and store in small aliquots of ~4X12" at -20 in 50 ml conical
Ice
Ice bucket
Kimwipe	Fisher Scientific	06-666A
Leica GZ4 StereoZoom microscope			for screening transgenic mosquitoes (if applicable)
Paintbrush	AIT synthetic brush	size 10-0	for manipulating larvae/pupae (Amazon)
Panty hose	Walmart	L'eggs Everyday	Women's Nylon Plus Knee Highs Sheer Toe, 16 pairs (plus fits the carton)
Pencils	Any brand
Plastic containers for 2° storage of cartons	Walmart		Sterilite 58 Qt Storage Box Clear Base White Lid Set of 8
Plastic containers for growing larvae	Walmart		Sterilite 28 Qt. Storage Box Plastic, White, Set of 10
Plastic containers for hatching larvae	Walmart		Sterilite 6 Qt. Storage Box Plastic, White
polypropylene clear deli containers	WebstaurantStore	127DM12BULK	12 oz, or 16 oz if needed for bigger (127RD16BULK)
Rubber bands	Office Max	#100736/#909606 /#3777415	12", #64 and #10
Rubber stopper	VWR	217-0515	for mosquito enclosement
Sugar source, such as sugar cubes or raisins
Tetramin flake food	Tetramin	16106
tpsDig	Stony Brook Morphometrics		A free morphometric image-processing software distributed online available at https://www.sbmorphometrics.org/
tpsUtil	Stony Brook Morphometrics		A free morphometric image-processing software distributed online available at https://www.sbmorphometrics.org/
White organza fabric 8” × 8”	FabricWholesale.com	4491676	Joann Casa Collection Organza Fabric by Casa Collection
Whitman Grade 1 Qualitative Filter paper	Whitman	1001-824	for egg papers. The white color makes it easier to see the black eggs.