MEH was funded by the HJ Van Cleave Professorship at the University of Illinois, Urbana-Champaign. In addition, for funding we thank the Human Frontier Science Program (to M.E.H. and T.G.) and the European Social Fund and the state budget of the Czech Republic, project no. CZ.1.07/2.3.00/30.0041 (to T.G.). We thank Ocean Optics for covering publication costs.

Ocean Optics has funded page charges for this manuscript.

Although egg rejection experiments are the most common method to study brood parasite-host coevolution70, concerted efforts to standardize materials, techniques, or protocols are lacking. This is especially problematic for meta-analyses. No meta-analysis, to our knowledge, of host egg rejection so far has controlled for methodological discrepancies among studies71,72, including what is considered mimetic or non-mimetic. This represents a major problem because mimetic (by human standards) eggs can be rejected by hosts more often than seemingly non-mimetic ones73, which shows that human color classification is both inadequate and inappropriate for inferences regarding avian cognition18. Additionally, meta-analyses ignore the fact that some studies, even of the same host species, handled desertions differently, either counting them as responses22 or excluding them from analyses7,71,72. Moreover, misclassifying egg models as either non-mimetic or mimetic eggs, can lead to fallacious comparisons between studies that both used these classifications for different egg model types74. Differences between studies1,74 may reflect both differences in the study design (e.g., model egg types) or differences between populations, both to an unknown degree; this prevents clear interpretation of the differences and precludes rejecting null and alternative hypotheses. This protocol provides a standardized approach for egg rejection experiments, and particularly emphasizes the coloring and quantifying the color of egg models. Following or adapting (and appropriately reporting) this protocol should promote the methodological standardization necessary for productive scientific debate, inter-study comparisons, and future progresses in this field of research.
Since eggshell coloration is determined by only two pigments, protoporphyrin IX appearing brown, and biliverdin IX&#945; appearing blue-green66,75,76, eggshell colors only occupy a small section of avian vision66. This variation can be replicated through carefully formulating acrylic paints that match natural eggshell colors, and this will lead to a better understanding of host recognition mechanisms. For example, Turdus hosts are more likely to reject brown eggs than blue-green eggs, despite the absolute perceived color difference between these foreign eggs and their own (Figure 2). Responses to eggs colored along color gradients vary considerably, highlighting the importance of accurately producing and reproducing the colors used in egg rejection experiments. Even variation in spot coloration can result in striking differences in host response (Figure 3)21. By using this approach, researchers can more systematically probe the limits of host recognition and uncover the relative importance of chromatic and luminance pathways in informing host decisions.
Despite the benefits that this approach provides for quantifying host responses across a phenotypic range, it is not suited for testing every hypothesis. When rejection rates are necessary for testing hypotheses, particularly for inter-population comparisons, using one or more consistent egg model types would be a less costly and demanding approach. For example, presenting specific egg model types representative of specific parasitic egg polymorphisms, can provide insight into historic and contemporary selection pressures77. Calculating rejection rates is impossible when each egg is a unique color; however, quantifying host responses across a range of potential parasitic egg colors can provide insight into questions related to decision thresholds and discrimination abilities. Specifically, this approach provides a tool for researchers to measure a host&#39;s egg discrimination abilities. This protocol outlines paint recipes to help standardize the color of egg models used for either approach. Moreover, regardless of the approach chosen, researchers should report the paints they used for coloring their egg models and should quantify those colors carefully. This should enhance inter-study comparisons and meta-analysis.
Egg rejection studies with continuous color, pattern, size, and/or shape traits have revolutionized the field of avian host-parasite arms-race studies in combination with the now standard usage of avian visual perception modelling73,78. There is now evidence that some hosts do not only use the absolute perceptual difference between own and foreign eggs in egg recognition, but instead base rejection decisions upon the direction of these difference along the phenotypic gradient of avian eggshell color9. Future research should use habituation/dishabituation or operant training studies to assess whether hosts of avian brood parasites can perceive and discriminate between natural and artificial egg colors in non-egg recognition contexts. Furthermore, these same experiments could reveal whether current-, historic-, and non-hosts can distinguish natural from artificial eggshell, which would highlight the role of sensory mechanisms in coevolutionary arms-races. Finally, adequately incorporating and replicating the UV components of avian egg coloration and maculation into egg rejection studies is a necessary challenge to overcome by future research79; this will be necessary to assess whether UV-based egg color signals represent a perceptually salient or unique cue for egg recognition and rejection in hosts of avian brood parasites. By using this consistent protocol, researchers can create new experiments and more easily interpret and compare their findings6,29,30,31.

Brood parasites lay their eggs in the nests of other species that may then raise their young and pay the costs associated with parental care1,2,3. This act of deception to outwit the host on the part of the parasite and sleuthing to detect the parasite on the part of the host provides strong selective pressures on both actors. In some cases of avian brood parasitism, the host&#39;s recognition of disparate parasitic eggs selects for parasites that mimic host eggs, which produces an evolutionary arms race between host and parasite4. Studying brood parasitism is important because it is a model system for investigating coevolutionary dynamics and decision-making in the wild5. Egg rejection experiments are one of the most common methods used for studying avian brood parasitism in the field and an important tool that ecologists use to investigate interspecific interactions6.
During the course of egg rejection experiments, researchers typically introduce natural or model eggs and assess the host&#39;s response to these experimental eggs over a standardized period. Such experiments can involve swapping real eggs (that vary in appearance) between nests7, or dyeing or painting the surfaces of real eggs (optionally adding patterns) and returning them to their original nests8, or generating model eggs that have manipulated traits such as color9, spotting10, size11, and/or shape12. The host response to eggs of varying appearance can provide valuable insight into the information content they use to reach an egg rejection decision13 and just how different that egg needs to be to elicit a response14. Optimal acceptance threshold theory15 states that hosts should balance the risks of mistakenly accepting a parasitic egg (acceptance error) or mistakenly removing their own egg (rejection error) by examining the difference between their own eggs (or an internal template of those eggs) and the parasitic eggs. As such, an acceptance threshold exists beyond which hosts decide a stimulus is too different to tolerate. When parasitism risk is low, the risk of acceptance errors is lower than when the risk of parasitism is high; thus, decisions are context specific and will shift appropriately as perceived risks change14,16,17.
Optimal acceptance threshold theory assumes that hosts base decisions upon continuous variation in host and parasite phenotypes. Therefore, measuring host responses to varying parasite phenotypes is necessary to establish how tolerant a host population (with its own phenotypic variation) is to a range of parasitic phenotypes. However, virtually all prior studies have relied on categorical egg color and maculation treatments (e.g., mimetic/non-mimetic). Only if host eggshell phenotypes do not vary, which is not a biologically practical expectation, would all responses be directly comparable (regardless of the degree of mimicry). Otherwise, a &#34;mimetic&#34; egg model will vary in how similar it is to host eggs within and between populations, which could potentially lead to confusion when comparing findings18. Theory suggests that host decisions are based upon the difference between the parasitic egg and their own14, not necessarily a particular parasitic egg color. Therefore, using a single egg model type is not an ideal approach to test hypotheses on host decision thresholds or discrimination abilities, unless the just noticeable difference (hereafter JND) between the egg model type and individual host egg color is the variable of interest. This also applies to experimental studies that swap or add natural eggs to test host responses to a natural range of colors19. However, while these studies do allow for variation in host and parasite phenotypes, they are limited by natural variation found in traits6, particularly when using conspecific eggs7.
By contrast, researchers that make artificial eggs of varied colors are free from the constraints of natural variation (e.g., they can investigate responses to superstimuli20), allowing them to probe the limits of host perception6. Recent research has used novel techniques to measure host responses across a phenotypic range, by painting experimental eggs designed to match and surpass the natural range of variation in eggshell9 and spot colors21. Studying host responses to eggs with colors along gradients can uncover underlying cognitive processes because theoretical predictions, such as acceptance thresholds15 or coevolved mimicry4, are based on continuous differences between traits. For example, by using this approach, Dainson et al.21 established that when chromatic contrast between eggshell ground coloration and spot coloration is higher, the American Robin Turdus migratorius tends to reject eggs more strongly. This finding provides valuable insights on how this host processes information, in this case through spotting, to decide whether to remove a parasitic egg. By customizing paint mixtures, researchers can precisely manipulate the similarity between an experimental egg&#39;s color and host&#39;s egg color, while standardizing other confounding factors such as spotting patterns10, egg size22 and egg shape23.
To encourage further replication and metareplication24 of classic and recent egg rejection work, it is important that scientists use methodologies that are standardized across phylogeny (different host species)7,22, space (different host populations)7,22,25,26 and time (different breeding seasons)7,22,25,26,27, which was done only rarely. Methodologies that were not standardized28 were later shown to lead to artefactual results29,30. This paper serves as a set of guidelines for researchers seeking to replicate this type of egg rejection experiment that examines responses to continuous variation and highlights a number of important methodological concepts: the importance of control nests, a priori hypotheses, metareplication, pseudoreplication, and color and spectral analysis. Despite egg rejection experiments dominating the field of avian host-parasite coevolution, no comprehensive protocol exists yet. Therefore, these guidelines will be a valuable resource to increase inter- and intra-lab repeatability as the true test of any hypothesis lies in metareplication, i.e., repeating whole studies across phylogeny, space and time24, which can only be meaningfully done when using consistent methods29,30,31.

<table><tbody><tr><td>Replicator Mini +</td><td>Makerbot</td><td></td><td></td></tr><tr><td>Professional Acrylic Paint Cobalt Turquoise Light</td><td>Winsor &amp;amp; Newton</td><td>28382</td><td></td></tr><tr><td>Professional Acrylic Paint Titanium White</td><td>Winsor &amp;amp; Newton</td><td>28489</td><td></td></tr><tr><td>Professional Acrylic Paint Cobalt Green</td><td>Winsor &amp;amp; Newton</td><td>28381</td><td></td></tr><tr><td>Professional Acrylic Paint Cobalt Turquoise</td><td>Winsor &amp;amp; Newton</td><td>28449</td><td></td></tr><tr><td>Professional Acrylic Paint Burnt Umber</td><td>Winsor &amp;amp; Newton</td><td>28433</td><td></td></tr><tr><td>Professional Acrylic Paint Red Iron Oxide</td><td>Winsor &amp;amp; Newton</td><td>28486</td><td></td></tr><tr><td>Professional Acrylic Paint Cadmium Orange</td><td>Winsor &amp;amp; Newton</td><td>28437</td><td></td></tr><tr><td>Professional Acrylic Paint Raw Umber Light</td><td>Winsor &amp;amp; Newton</td><td>28391</td><td></td></tr><tr><td>Professional Acrylic Paint Yellow Ochre</td><td>Winsor &amp;amp; Newton</td><td>28491</td><td></td></tr><tr><td>Professional Acrylic Paint Mars Black</td><td>Winsor &amp;amp; Newton</td><td>28460</td><td></td></tr><tr><td>Paint Brush</td><td>Utrecht</td><td>206-FB</td><td>Filbert brush</td></tr><tr><td>Paint Brush</td><td>Utrecht</td><td>206-F</td><td>Flat brush</td></tr><tr><td>Hair Dryer</td><td>Oster</td><td>202</td><td></td></tr><tr><td>Fiber optic cables</td><td>Ocean Optics Inc.</td><td>OCF-103813</td><td>1 m custom bifurcating fiber optic assembly with blue zip tube (PVDF), 3.8mm nominal OD jeacketing and 2 legs</td></tr><tr><td>Spectrometer</td><td>Ocean Optics Inc.</td><td>Jaz</td><td>Spectrometer unit with a 50 um slit width, installed with a 200-850 nm detector (DET2B-200-850), and grating option # 2.</td></tr><tr><td>Battery and SD card module for spectrometer</td><td>Ocean Optics Inc.</td><td>Jaz-B</td><td></td></tr><tr><td>Light source</td><td>Ocean Optics Inc.</td><td>Jaz-PX</td><td>A pulsed xenon light source</td></tr><tr><td>White standard</td><td>Ocean Optics Inc.</td><td>WS-1-SL</td><td>made from Spectralon</td></tr><tr><td>OHAUS Adventurer Pro Scale</td><td>OHAUS</td><td>AV114C</td><td>A precision microbalance</td></tr><tr><td>Gemini-20 portable scale</td><td>AWS</td><td>Gemini-20</td><td>A standard scale</td></tr><tr><td>Empty Aluminum Paint Tubes (22 ml)</td><td>Creative Mark</td><td>NA</td><td></td></tr><tr><td>Telescopic mirror</td><td>SE</td><td>8014TM</td><td></td></tr><tr><td>GPS</td><td>Garmin</td><td>Oregon 600</td><td></td></tr><tr><td>220-grit sandpaper</td><td>3M</td><td>21220-SBP-15</td><td>very fine sandpaper</td></tr><tr><td>400-grit sandpaper</td><td>3M</td><td>20400-SBP-5</td><td>very fine sandpaper</td></tr><tr><td>color analysis software: &amp;lsquo;pavo&amp;rsquo;, an R package</td><td>for use in, &lt;em&gt;R: A language and environment for statistical computing&lt;/em&gt;</td><td>v 1.3.1</td><td>https://cran.r-project.org/web/packages/pavo/index.html</td></tr><tr><td>UV clear transparent</td><td>Flock off!</td><td>UV-001</td><td>A transparent ultraviolet paint</td></tr><tr><td>Plastic sandwich bags</td><td>Ziploc</td><td></td><td>Regular plastic sandwich bags from Ziploc that can be purchased at the supermarket.</td></tr><tr><td>Kimwipes</td><td>Kimberly-Clark Professional</td><td>34120</td><td>11 x 21 cm kimwipes</td></tr><tr><td>Toothbrush</td><td>Colgate</td><td></td><td>Toothbrush</td></tr></tbody></table>

probing the limits of egg recognition using egg rejection experiments along phenotypic gradients

Brood parasites lay their eggs in other females' nests, leaving the host parents to hatch and rear their young. Studying how brood parasites manipulate hosts into raising their young and how hosts detect parasitism provide important insights in the field of coevolutionary biology. Brood parasites, such as cuckoos and cowbirds, gain an evolutionary advantage because they do not have to pay the costs of rearing their own young. However, these costs select for host defenses against all developmental stages of parasites, including eggs, their young, and adults. Egg rejection experiments are the most common method used to study host defenses. During these experiments, a researcher places an experimental egg in a host nest and monitors how hosts respond. Color is often manipulated, and the expectation is that the likelihood of egg discrimination and the degree of dissimilarity between the host and experimental egg are positively related. This paper serves as a guide for conducting egg rejection experiments from describing methods for creating consistent egg colors to analyzing the findings of such experiments. Special attention is given to a new method involving uniquely colored eggs along color gradients that has the potential to explore color biases in host recognition. Without standardization, it is not possible to compare findings between studies in a meaningful way; a standard protocol within this field will allow for increasingly accurate and comparable results for further experiments.

Generating colorful egg models
Reflectance spectra of custom paint mixtures and natural eggs are shown in Figure&#160;1A-1D. Paint mixtures used in brood parasitism studies should closely correspond with natural reflectance measurements in terms of spectral shape (color) and magnitude (brightness). If that is achieved, the color of the experimental egg should be perceived by the host as a natural egg color. To assess host recognition, these reflectances should be transformed into a relevant avian color space. To do this, the product of these reflectance spectra, solar irradiance, and photoreceptor sensitivity can be integrated to calculate quantum catches37. Avian color perception is different than in humans, because birds see colors using four, rather than three photoreceptors. The quantum catch from these four receptors can be transformed into coordinates within a tetrahedral color space (Figure 1E), where each vertex represents the relative stimulation of a specific photoreceptor: ultraviolet, short, medium, and long wavelength-sensitive photoreceptors41. Coordinates within this space provide a method for comparing colors and phenotypic diversity of colors, which is relatively limited with respect to the color of birds eggshells66. Plotting experimental egg color in a color space is important because their coordinates within that space will determine if these eggs would appear natural. Figure 1 illustrates the colors of custom paints (colorful dots, letters correspond with the reflectance spectra shown in Figure&#160;1A-1D) that are described in this paper and how they compare to natural birds&#39; eggshell colors. This novel approach provides opportunities for novel experimental designs and can provide new insights into host egg recognition.
Quantifying host responses to eggshell colors
Explicitly or implicitly, all but one9 previous study has assumed that hosts respond to trait dissimilarity, e.g., the difference between a parasitic egg and their own, in an absolute or symmetrical fashion (Figure 2, dashed). This difference usually varies from identical (0) to infinitely different; however, most traits vary along multiple dimensions and there is no a priori reason to assume their responses should be similar across the phenotypic space. Research that manipulates traits across their full phenotypic range (Figure 2, solid), can test this assumption. For two Turdus spp., Hanley et al.9 found that between a brown and blue-green egg, both equally different from the host&#39;s own, the brown egg was more likely to be rejected. By using egg models with known coordinates within a standard avian color space (Figure 1E), researchers can work within the natural phenotypic range or extend that phenotypic range (e.g., towards blue-green or brown) to explore host response and probe the perceptual limits of host recognition. Such an approach provides a context (based on phenotypic space) to understand hosts&#39; responses.
Quantifying host responses to eggshell spot colors
A recent study21 showed that American robins are more likely to reject spotted experimental eggs when they perceive those spots as browner than the blue-green color of the eggshell (Figure 3). This host lays unspotted eggs, but their parasite the brown-headed cowbird Molothrus ater has brown spots and therefore, this decision rule seems adaptive. Such findings reinforce previous findings that have shown that American robins base decisions on both ground color and spots13; however, by measuring their responses across a color gradient Dainson et al. were able to establish that American robins use the chromatic contrast between ground and spot color in egg rejection decisions21. Experimental designs employing such continuous variation in coloration enable researchers to explore the role of host sensory and perceptual mechanisms in egg recognition more thoroughly.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57512/57512fig1v2.jpg" /> 
Figure 1. Representative variation in natural avian and artificial eggshell colors.&#160;The average of ten spectral reflectance measurements of (A) the blue-green, (B) beige, (C) brown, (D) white, and (E) dark brown paint mixtures (1.2.1 to 1.2.5, solid lines) alongside the reflectance of a real egg with a similar appearance: (A) American robin T. migratorius, (B) quail C. japonica, a (C) brown and (D) white domestic chicken egg Gallus g. domesticus (dashed lines). The peak in ultraviolet reflectance in (D) is due to the removal of the cuticle67. Inset photographs of real eggs on the left and artificial eggs to the left to scale (the bar above &#8220;artificial&#8221; represents 1 cm). The image of the real quail egg (inset B) was modified from a photograph taken by <a href="https://commons.wikimedia.org/wiki/File:Coturnix_coturnix_confisa_MHNT.ZOO.2010.11.7.1.jpg">Roger Culos</a> that is licensed under <a href="https://creativecommons.org/licenses/by-sa/4.0/deed.en">CC BY 4.0</a>. We illustrate adult birds as inset images (photo credits of bird insets A-D respectively: Tom&#225;&#353; Grim, <a href="https://commons.wikimedia.org/wiki/File:Japanese_Quail.jpg">Ingrid Taylar</a> under <a href="https://creativecommons.org/licenses/by/2.0">CC BY 2.0</a>, <a href="https://commons.wikimedia.org/wiki/File:Rhode_Island_Red.jpg">Sherool</a>, and <a href="https://commons.wikimedia.org/wiki/File:Leghorn_chicken.jpeg">Dejungen</a> under <a href="https://creativecommons.org/licenses/by-sa/3.0/">CC-BY-SA-3.0</a>). The avian perceived colors are also plotted within the (E) avian tetrahedral color space for the average ultraviolet-sensitive avian viewer. The vertices represent the relative stimulation of the ultraviolet (U), short (S), medium (M), and long (L) wavelength-sensitive photoreceptors. Gray dots represent the colors of natural avian eggs across the full phylogenetic diversity66, from previously published data68, while the colorful dots represent the colors of custom paint formulations here (steps 1.1.1 to 1.2), and small solid dots represent intermediate colors (step 1.3). Italic letters beside colorful dots reference spectral reflectances shown in this figure, while (e) references the dark spots from a quail egg.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57512/57512fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57512/57512fig2v2.jpg" /> 
Figure 2. Representative host egg rejections of eggs with variable eggshell coloration. Traditionally, the predicted (dashed) rejection probability for a host is based upon the absolute perceived difference between the hosts&#39; egg and foreign egg (i.e., as the foreign egg is more different responses to that egg are more likely, no matter the direction of the difference in the color space). This practice ignores natural variation in the host&#39;s own egg color. However, it is more likely that American robins (N = 52) will reject brown eggs than equally dissimilar blue-green eggs (solid line), which highlights the importance of examining host responses across a phenotypic gradient9.This figure was modified from Hanley et al.9 and these data69 are licensed under CC BY 4.0.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57512/57512fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57512/57512fig3v2.jpg" /> 
Figure 3. Representative host egg rejection of eggs with variable spot coloration. The chromatic contrast (JND) between the spot colors painted on experimental model eggs and the ground color of these models predicted host response (0 = acceptance, 1 = ejection) in the American robin. This figure has been modified from Dainson et al.21.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57512/57512fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Probing the Limits of Egg Recognition Using Egg Rejection Experiments Along Phenotypic Gradients at JoVE.com

Probing the Limits of Egg Recognition Using Egg Rejection Experiments Along Phenotypic Gradients

This protocol provides guidelines for running egg rejection experiments: outlining techniques for painting experimental egg models to emulate the colors of natural bird eggs, conducting fieldwork, and analyzing the collected data. This protocol provides a uniform method for conducting comparable egg rejection experiments.

Brood parasites lay their eggs in other females' nests, leaving the host parents to hatch and rear their young. Studying how brood parasites manipulate ...

probing-limits-egg-recognition-using-egg-rejection-experiments-along

Nanyang Technological University

Research

JoVE Journal

Behavior

8.2K Views.  Long Island University-Post. This protocol provides guidelines for running egg rejection experiments: outlining techniques for painting experimental egg models to emulate the colors of natural bird eggs, conducting fieldwork, and analyzing the collected data. This protocol provides a uniform method for conducting comparable egg rejection experiments.

Video: Probing the Limits of Egg Recognition Using Egg Rejection Experiments Along Phenotypic Gradients

Upright Imaging of Drosophila Egg Chambers

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective.

Upright Imaging of Drosophila Egg Chambers

The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in ...

Developmental Biology

Functional Cloning Using a Xenopus Oocyte Expression System

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.

Functional Cloning Using a Xenopus Oocyte Expression System

We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in ...

Windowing Chicken Eggs for Developmental Studies

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling.  In addition, it allows for molecular perturbations of several types, including placement of protein-coated beads and introduction of plasmid DNA using in ovo electroporation.  These experiments have yielded important data on the development of the central and peripheral nervous systems.  For many of these studies, it is necessary to open the eggshell and reclose it without perturbing the embryo's growth.  The embryo can be examined at successive developmental stages by re-opening the eggshell.  While there are several excellent methods for opening chicken eggs, in this article we demonstrate one method that has been optimized for long survival times.  In this method, the egg rests on its side and a small window is cut in the shell.  After the experimental procedure, the shell is used to cover the egg for the duration of its development.  Clear plastic tape overlying the eggshell protects the embryo and helps retain hydration during the remainder of the incubation period.  This method has been used beginning at two days of incubation and has allowed survival through mature embryonic ages.

The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling. In this article we demonstrate one egg preparation method that has been optimized for long survival times.

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has ...

Biology

Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental studies. Like other aphids, A. pisum propagate rapidly via parthenogenetic viviparous reproduction, where the embryos develop within egg chambers in an assembly-line fashion in the ovariole. Previously we have established a robust platform of whole-mount in situ hybridization allowing detection of mRNA expression in the aphid embryos. For analyzing the expression of protein, though, established protocols for immunostaining the ovarioles of asexual viviparous aphids did not produce satisfactory results. Here we report conditions optimized for increasing tissue permeability and decreasing background staining, both of which were problems when applying established approaches. Optimizations include: (1) incubation of proteinase K (1 &#181;g/ml, 10 min), which was found essential for antibody penetration in mid- and late-stage aphid embryos; (2) replacement of normal goat serum/bovine serum albumin with a blocking reagent supplied by a Digoxigenin (DIG)-based buffer set and (3) application of methanol rather hydrogen peroxide (H2O2) for bleaching endogenous peroxidase; which significantly reduced the background staining in the aphid tissues. These critical conditions optimized for immunostaining will allow effective detection of gene products in the embryos of A. pisum and other aphids.

A protocol of whole-mount immunostaining on aphid embryos is presented, in which critical conditions for decreasing background staining and increasing tissue permeability are addressed. These conditions are specifically developed for effective detection of protein expression in the embryonic tissues of aphids.

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental ...

Injecting Gryllus bimaculatus Eggs

Altering gene function in a developing organism is central to different kinds of experiments. While tremendously powerful genetic tools have been developed in traditional model systems, it is difficult to manipulate genes or messenger RNA (mRNA) in most other organisms. At the same time, evolutionary and comparative approaches rely on an exploration of gene function in many different species, necessitating the development and adaptation of techniques for manipulating expression outside currently genetically tractable species. This protocol describes a method for injecting reagents into cricket eggs to assay the effects of a given manipulation on embryonic or larval development. Instructions for how to collect and inject eggs with beveled needles are described. This relatively straightforward technique is flexible and potentially adaptable to other insects. One can gather and inject dozens of eggs in a single experiment, and survival rates for buffer-only injections improve with practice and can be as high as 80%. This technique will support several types of experimental approaches including injection of pharmacological agents, in vitro capped mRNA to express genes of interest, double-stranded RNA (dsRNA) to achieve RNA interference, use of clustered regularly interspaced short palindromic repeats (CRISPR) in concert with CRISPR-associated protein 9 (Cas9) reagents for genomic modification, and transposable elements to generate transient or stable transgenic lines.

Injecting Gryllus bimaculatus Eggs

Here we present a protocol to inject cricket eggs, a technique which serves as a foundational method in many experiments in the cricket, including, but not limited to, RNA interference and genomic manipulation.

Altering gene function in a developing organism is central to different kinds of experiments. While tremendously powerful genetic tools have been ...

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the &#34;greater of two goods.&#34; However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

Neuroscience

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which enables them to seek out their preferred thermal landscape. To define the temperature preferences of larvae over a defined linear range, we developed an assay using a temperature gradient. To establish a single-directional gradient, two aluminum blocks are connected to independent water baths, each of which controls the temperature of individual blocks. The two blocks set the lower and upper limits of the gradient. The temperature gradient is established by placing an agarose-coated aluminum plate over the two water-controlled blocks so that the plate spans the distance between them. The ends of the aluminum plate that is set on the top of the water blocks defines the minimum and maximum temperatures, and the regions in-between the two blocks form a linear temperature gradient. The gradient assay can be applied to&#160;larvae of different ages and can be used to identify mutants that exhibit phenotypes, such as those with mutations affecting genes encoding TRP channels and opsins, which are required for temperature discrimination.

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Here, we present a protocol to determine the preferred environmental temperature of Drosophila larvae using a continuous thermal gradient.

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...

Mating and Egg Staging: A Method to Generate Embryos and Sort Them by Developmental Stage

Source: Hostelley T.L., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/56187/" target="_blank">Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish</a>. J. Vis. Exp. (2017)
The article describes a protocol to generate zebrafish embryos through natural mating and sorting their developmental stages. The protocol is used further for analysis of pancreatic &#946;-cells by using transgenic fishes.

Krycie i ocena stopnia zaawansowania jaj: metoda generowania zarodków i sortowania ich według etapu rozwoju

Source: Hostelley T.L., et al. Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish. J. Vis. Exp. (2017)
The article ...

JoVE Encyclopedia of Experiments

Danio rerio (zebrafish)

Embryo

Drosophila Egg Collection and Dechorionation: A Method to Remove the Outermost Egg Layer

Source: Koyle, M. L., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/54219/" target="_blank">Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions</a>. J. Vis. Exp. (2016).
The Drosophila embryo is surrounded by protective outer membranes. To gain access to the embryo, the chorion&#8211;the outermost of the membranes&#8211;is often removed. This video describes a commonly used method of dechorionation using sodium hypochlorite. The example protocol demonstrates the procedure while employing sterile techniques necessary for axenic or gnotobiotic animal rearing.

Drosophila Zbieranie jaj i dechorionacja: metoda usuwania najbardziej zewnętrznej warstwy jaj

Source: Koyle, M. L., et al. Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions. J. Vis. Exp. (2016).
The Drosophila ...

Drosophila melanogaster (fruit fly)

Egg Laying Assay: A Method to Quantify the Egg-Laying Behavior of C. elegans

Source: Lee, S. Y. and Kang, K. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/60419/measuring-effects-bacteria-chemicals-on-intestinal-permeability">Measuring the Effect of Chemicals on the Growth and Reproduction of&#160;Caenorhabditis elegans</a>.&#160;J. Vis. Exp. (2017).
C. elegans egg-laying behavior can provide important information in developmental, reproductive, and anatomical studies.&#160; This video introduces the important steps of an egg laying assay and shows a sample experiment in which a drug is tested for reproductive toxicity.

Test składania jaj: metoda ilościowego określania zachowania C. elegans podczas składania jaj

Source: Lee, S. Y. and Kang, K. Measuring the Effect of Chemicals on the Growth and Reproduction of&#160;Caenorhabditis elegans.&#160;J. Vis. Exp. 
...

Probing the Limits of Egg Recognition Using Egg Rejection Experiments Along Phenotypic Gradients

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High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Krycie i ocena stopnia zaawansowania jaj: metoda generowania zarodków i sortowania ich według etapu rozwoju

Drosophila Zbieranie jaj i dechorionacja: metoda usuwania najbardziej zewnętrznej warstwy jaj

Test składania jaj: metoda ilościowego określania zachowania C. elegans podczas składania jaj