Killifish inhabit brackish and fresh waters in the Americas, Europe, Asia and Africa. These oviparous cypriniform fishes are often brightly colored, highly active and typically measure 2.5-5 cm long, but range from 8 mm to 35 centimeters at maturity. Many species of killifish inhabit permanent streams, rivers and lakes and live for up to three years, but some more species of killifish inhabit vernal pools that last for only a few days, weeks or months before drying up completely. These annual killifishes risk extinction, due to habitat loss, restricted distribution, and low dispersal of most species, in countries like Brazil [6]. The killifish rely on diapause as an integral part of their breeding cycles. Diapause, a physiological state of dormancy, occurs in the egg stage after laying and is characterized by slowed metabolism, decreased heart rate and reduced oxygen consumption [7] and is regulated through temperature-dependent vitamin D signaling [8]. These survival mechanisms are echoed in other species of killifish through evolution and speciation events as some adult killifish can survive out of water for several weeks and others can survive extreme cold and anoxia [9, 10].

Killifish have been used as model organisms for over a decade. Some species of killifish display high resistance to environmental toxins that developed quickly over the last 50-60 years. Their resistance to toxins has made them appealing to researchers who seek answers to surviving in an increasingly polluted planet and in harsh environmental conditions. For example, Oziolor EM et al identifed an introgressed aryl hydrocarbon receptor locus in Gulf killifish (Fundulus grandis) from Atlantic killifish (F. heteroclitus) that enhanced the survival of Gulf killifish in polluted habitats [25]. Short-lived species inhabiting ephemeral ponds attracted attention from scientists who seek clues to metabolism and aging [26-28].

The short lifespans of the killifish relative to mammals made them ideal for research. Their fecundity, short gestation, prolonged incubation of eggs, minimal space and energy requirements and trivial maintenance makes them ideal for the sterile conditions of the laboratory. The genes of these animals can also be easily manipulated [1], and eggs can be safely stored, transported or used immediately. Medium-throughput assays developed for whole organisms can be easily applied to the products of these processes [29].

Mammalian models are more likely to call research into more intense scrutiny by internal review boards (IRBs), and their maintenance and care are much more involved than fish models. Killifish are low-maintenance and can be used to study disease and other processes in either the whole fish or in organ systems. Sexually mature as early as 17 days from hatching, female killifish can lay between 10 and 20 eggs a day with a recorded maximum of 100 [30]. Killifish eggs can be incubated for as little as six weeks or as long as eight months depending on the species and storage conditions. This might seem like a drawback relative to mice (about 20 days gestation) and rats (up to 25 days), but the number of eggs means that many batches from a single spawning event can be revived at any time during that incubation period for experimentation. Research has also suggested that modeling human diseases in rodents robs researchers of insight unique to human cells; however, tissue cultures can involve more maintenance at a higher cost than fish models [31]. Waste products from killifish are also more environmentally friendly; dirty water can usually be discarded into the sanitary sewer. Killifish can be used alongside or as alternatives to other conventional models like those discussed above [32].

Figure 1. CRISPR/Cas9 gene modification in killifish. A. DNA oligos complementary to the target sequence are used to make gRNA. B. These gRNAs are injected into the fertilized egg (measures ~1mm) along with Cas9 mRNA after it has been embedded in an agarose trench or in low melt agar. Injection of phenol red dye helps visualize the blastodisc. C. Fish mature after approximately 20 days and are available for optional backcross [1].

All these features make killifish an excellent option for researchers seeking to study neurogenesis [22], metabolism, biogerontology and evolutionary adaptations to pollution and harsh climate.

There are more than 1270 species of killifish, but only a few are used in laboratory research. Typically, killifish used for research are either wild caught for population and environmental studies or are specific strains that are exchanged between laboratories. Some of the more commonly studied strains are listed in Table 1 along with relevant statistics about husbandry.

Figure 2. Construct for Tet-ON promoter. A. Tetracycline binds to the translated rtTA protein. B. rtTA binds to the Tet-op promoter resulting in transcription of the gene of interest. Binding only occurs in the presence of the tetracycline, though with some constructs, leaky transcription may occur. Figure adapted from [2].

Annual killifish are typically used to study metabolism, mitochondrial metabolism and physiological responses to stress conditions due to their remarkable tolerance for low oxygen conditions. Mummichog also has a high tolerance for stressful environmental conditions like anoxia, hypoxia, larger temperature fluctuations and changes in salinity. These qualities make mummichog useful for studies in metabolism, physiological responses to stress and toxins and even the plasticity of their mitochondrial genomes. Mangrove killifish are typically used to study evolution and are amphibious and hermaphroditic. Similarly, the Trinidadian killifish are also used for studies in evolution and behavior. Trinidadian killifish are usually studied in the wild or are wild-caught and studied in the lab.

In contrast, turquoise killifish are used to study aging and age-related diseases [33]. Their short lifespans and ease of husbandry make them ideal for this area of research. Medaka has remarkably small genomes, and many lines of inbred strains are available. These strains are used to study neurotoxicology, ecotoxicology, carcinogenesis, and disruption of endocrine systems [34-36].

Some of these strains have been studied extensively. Medaka, the Japanese killifish, has been subjected to systematic target-selected mutagenesis to produce a library of gene knockout models [37]. In addition to broad bodies of literature, searchable genomes have been published for mummichog, medaka and the turquoise killifish [3, 38-40].

Typically, these animals can be kept in small containers like plastic cups, though some more sophisticated setups include automated filtration. Breeding typically requires larger setups. Automatic water filters reduce the amount of time required for maintenance for a nominal initial investment. Except for hermaphroditic species, breeding pairs are kept together overnight in a container where each fish is separated by a barrier that allows each animal to see and share water with the other. After several hours of introduction, the barrier is removed, and the two interact directly. Females spawn either on a mop of dark-colored acrylic yarn or peat moss. Both of these materials are easy to sterilize beforehand and allow easy visualization of the eggs. It can be difficult to enumerate the eggs without removing each one from the substrate, but this step makes desiccation before incubation much easier [41].

Figure 3. In vitro CRISPR/Cas9 gRNA synthesis. A. DNA oligos are hybridized to ligated genomic target sequences for amplification. B. Targets are amplified via PCR. C. Complimentary DNA sequences are purified and ready for amplification using T7 RNA polymerase. D. Resulting cleaned products are ready for injection with Cas9 mRNA. Figure adapted from [3].

Many of the techniques used for injecting chemical and genetic materials into killifish eggs are adapted from another popular vertebrate model, zebrafish (Danio rerio). Microinjection techniques using glass pipettes deliver materials through the sturdy chorion of killifish eggs (Figure 1). Killifish eggs have a tough chorion relative to zebrafish so a shorter needle can be used to inject materials into the egg, as described by Hartmann et al Typically, materials are injected into the blastodisc as it divides and backcross is performed later for a clean strain. If a chimeric animal is desired, then materials are injected into specific cells in the dividing blastosphere. Allografting in adult animals should take into account circadian rhythms affecting possible immune responses as shown by Nevid et al [42-44].

CRISPR/cas9 is popular for manipulating genes in killifish because it allows targeted insertion and knock-out of genes. In this method, carefully designed RNA that reflect the changes a researcher wishes to make to the killifish genome (gRNA) are injected along with RNA encoding the machinery necessary for altering the DNA. Harel et al have described these techniques in great detail, but several papers have been written describing the process [1, 3, 20].

Figure 4. Example of fluorescent antibody use and microscopy in mummichog killifish (Fundulus heteroclitus). These ionocytes show the distribution of cystic fibrosis transmembrane conductance regulator (CFTR) and with no lysine kinase (WNK1). Top figure: This image shows an x-z scan; it is a side view of a saltwater ionocyte (ic) with a stained accessory cell (ac). The white bar shows 5 µm for scale. The CFTR (A) is present in the apical crypt (ap), while the rest of the ionocyte and the accessory cells stained strongly for WNK1 phosphoantibody (B and C). Bottom figure: A drawing that models the distribution CFTR and WNK1 distribution in mummichog ionocytes (n: nucleus, pvc: pavement cell layer). Description adapted from correspondence with Regina Cozzi who generously supplied her images. Her work is conducted with WS Marshall and is funded by NSERC grant RGPIN 3698-2000.

In order for materials to be injected into the killifish eggs, eggs must be stabilized. Killifish eggs can either be bound in trenches molded into conventional agar or by embedding the eggs in low-melting temperature agar. Delicate zebrafish embryos are usually secured in an agarose trench for injection and this technique can also be used for killifish eggs, but embedding killifish eggs in low-melt agarose did not affect the viability of the embryos according to data [50]. Depending on when and where the embryos are injected, researchers have control over how and when genes of interest are expressed [50].

A broad variety of conditional and constitutive gene regulation are available for killifish. Conditional in vivo gene regulation using Tet-ON as shown in Figure 2, Cre/loxP, Gal4 upstream activation sequence and laser have been described as alternatives to heat-shock for localized gene expression and either have or will be adopted for use in killifish [2]. Colored fluorescent proteins have also been described for use in tracking protein expression in vivo in killifish [51].

Genetic manipulation techniques using directed and random mutagenesis are available using now commonly available tools like CRISPR or transposons as shown in Figure 3 [52]. Harel et al recommend an online tool, CHOPCHOP (https://chopchop.rc.fas.harvard.edu), to predict gRNA targets though many vendors offer similar tools. These software programs use genomic data to predict targets that include the necessary 3-nucleotide sequence motif (NNG) on the gRNA and the protospacer adjustment motif (PAM) downstream in the target DNA. These allow the Cas9 protein to bind and alter DNA as desired and designed by researchers.

Figure 5. qPCR as a method for assessing DNA damage in killifish. A. Animals or eggs can be treated with chemicals, radiation, conditional gene expression. B. DNA can be collected at desired time points or in single batches and quantified. C. qPCR and subsequent quantification via determination of densitometry in agarose after electrophoresis. D - E. Lesion frequency calculation f(x)=e-λ λ / X ! Poisson expression. Zero Class: f(0)=e- λ; λ – lesion frequency; AD = amplification of damaged template; AO = Amplification of non-damaged template; Lesion frequency / genomic strand: λ = -ln AD / AO. Figure adapted from [4, 5].

Transposon-mediated transgenesis is a similarly successful method for manipulating killifish genes as described [52]. Cao C et al co-injected a transgenic vector and transposase mRNA into one-cell-stage N. furzeri embryos to generate transgenic lines [53]. Table 2 lists methods for gene modification described for use in fish vertebrate models. All of these techniques offer a robust and almost universally accepted method for studying changes in homeostasis including the addition of human genes [1, 52].

Once genes have been modified, researchers must track and quantify the relevant phenomena. Because many varieties of carefully constructed colored fluorescent proteins are available for use in laboratory experiments [54], there are many opportunities to track gene expression in vivo. The insertional mutagenesis systems described in Table 2 allow researchers to add colored tags and subsequently track gene expression during development or ageing and under stress conditions. This will enable researchers to monitor protein expression in vivo and throughout the organism's entire lifespan [4, 5, 5, 54-56]. The short lifespan, along with the presence of physiological systems unique to the vertebrate, such as adaptive immune system or the hypothalamic‐pituitary axis, enable the killifish to be an excellent model for aging research [57].

Disrupting gene function is a valuable technique for studying aging and other phenomena in killifish. Typically, the aforementioned methods for manipulating genes are used, but when metabolism is more relevant sometimes methods for activating inserted genes without hormones, sugars or other materials that might unduly influence metabolism are more appropriate. Radiation, non-hormonal oxidative stressors and other chemicals can be used in these studies [28]. Table 2 lists several non-genetic ways to interfere with biological processes in killifish. Perturbing homeostasis without the potential of a confounding biological variable can yield unique insight into biological processes [4, 5, 58].

Fluorescent dyes can also be used to visualize cells either on their own or using antibodies. Confocal and fluorescent microscopy can create stunning 3-dimensional models and informative images as shown in Figure 4. These cells were stained with AlexaFluor secondary antibodies after being fixed, but there are many available dyes and stains [59].

DNA damage can also be tracked using a quantitative PCR (qPCR) protocol adapted for use in killifish [5]. This technique offers a quantitative assay for multiple tissue types after exposure to chemical mutagens and radiation. DNA from treated animals is collected, quantified, subjected to qPCR to assess DNA damage and then analyzed using Poisson distribution as shown in Figure 5 [4, 5, 55]. This technique allows researchers to precisely monitor DNA damage in real time.