We thank Mr. Taku Shimada, the delegate of AntRoom, Tokyo, Japan, for providing us with his photograph of V. emeryi reproductives. This project was funded by the Japan Society for the Promotion of Science (JSPS) Research Fellowship for Young Scientists (16J00011), and Grant in Aid for Young Scientists (B)(16K18626).

1. Field Collection and Maintenance of V. Emeryi Colonies in the Laboratory
NOTE: Nests of V. emeryi are found in rotting logs and fallen decaying tree branchesin secondary forests throughout Japan. This species shows two types of colonies, i.e., (1) colonies producing only long-winged queens and (2) colonies mainly producing short-winged queens in addition to small number of long-winged queens8,14. In this protocol, we collected the latter type of colonies in Ishikawa prefecture, Japan.
<ol>
	<li>Collect V. emeryi colonies during early summer. 
	NOTE: To obtain sufficient numbers of sexual individuals during the reproductive season, colonies containing more than 300 individuals are preferred.</li>
	<li>Transfer the ant specimens from the collected branches to an artificial plaster nest with a glass cover using an aspirator (Figure 2, left).</li>
	<li>Maintain colonies in the artificial nest at 25 &#176;C under a 16:8 h light/dark cycle. Provide tap water with a wash bottle to wet the plaster.
	<ol>
		<li>Add about 100 mg of dry cricket powder wrapped in aluminum foil and a brown sugar water-filled tip (20 &#181;L tip) every other day until new reproductives (F1 winged-queens and F1 males) emerge.</li>
	</ol>
	</li>
</ol>
2. Experimental Laboratory Crosses
NOTE: New reproductives start to emerge from late summer to autumn (Figure 3). Long-winged queens are produced sexually, and short-winged queens are produced clonally and have the maternal genome (Figure 1). Use long-winged queens and males for inbreeding crosses.
<ol>
	<li>To stop individuals from moving, place colonies in a constant environment room at 4 &#176;C for 15 min.</li>
	<li>Remove the mid-legs of 30 workers using forceps under a stereoscopic microscope and transfer them into new smaller plaster nest (Figure 2, right) for inbreeding crosses. 
	NOTE: The legs are removed to distinguish the present workers from the workers that will be produced by the subsequent inbreeding crosses.</li>
	<li>Add 3-4 larvae or pupae into a plaster nest containing workers. 
	NOTE: Workers show exploratory activity in the F0 queens-less colony. Larvae or pupae can effectively attract these workers and new reproductives in the center of the colony during crossing test. As a result, keep conditions of the experimental colony close to the normal colony.</li>
	<li>Transfer a long-winged queen and a male into a plaster nest prepared in step 2.3 for inbreeding crosses.</li>
	<li>Keep colonies at 25 &#176;C under a 16:8 h light/dark cycle with food and water provided as described in 1.3 until the queen lose her wings and lay eggs. 
	NOTE: This takes one week to a month.</li>
	<li>Check the experimental colony everyday under a stereoscopic microscope. After performing inbreeding crosses between the F1 offspring, eggs can be observed under a stereoscopic microscope.</li>
	<li>After the F1 queen starts laying eggs, remove F1 males and larvae or pupae added in step 2.3 from the nest to avoid mixing of the F1 generation (males and females used for inbreeding crosses) and the F2 generation (offspring produced from inbreeding crosses). 
	NOTE: If there are few males in the colony, it is possible to induce inbreeding crosses using one male and 1 to 3 queens in the same experimental colony.</li>
	<li>Keep colonies under the same conditionsas described in 1.3, until F2 offspring emerge. 
	NOTE: Transfer F1 queen and F2 offspring into new larger plaster nest (Figure 2, left) for long-term colony keeping.</li>
</ol>
3. Evaluation of Inbreeding Success
<ol>
	<li>DNA extraction and genotyping of the parental generation (F0)
	<ol>
		<li>Remove one leg of a F0 queen using forceps and transfer the leg to a 1.5 mL microtube containing 100 &#181;L of chelation agent.</li>
		<li>Under a stereoscopic microscope, dissect a female abdomen in glass dish filled with 300 &#181;L of ultrapure water using forceps and isolate the spermatheca containing the sperm from mated males.</li>
		<li>Peel away the tissue of the spermatheca and isolate the sperm from the tissue of the female using insect pins. 
		NOTE: To facilitate sperm extraction from the spermatheca, store female specimens in 100% EtOH for more than one day before dissection.</li>
		<li>Using a micropipette, transfer the sperm into a 1.5 mL microtube containing 100 &#181;L of chelation agent.</li>
		<li>Incubate samples of F0 queen and sperms prepared in step 3.1.1 and 3.1.3, respectively, at 95 &#176;C for 20 min. Flash centrifuge the microtube and store at 4 &#176;C.</li>
		<li>Genotype all samples using method described elsewhere4.</li>
	</ol>
	</li>
	<li>DNA extraction from the pair of ants used for inbreeding crosses (F1)
	<ol>
		<li>After confirming egg production by the sib mated F1 queen, extract the DNA of the queen using her shed wings or one mid-leg and genotype using same method described in section 3.1 above.</li>
		<li>Extract DNA of F1 male using one leg and genotype them using same method described in section 3.1 above. 
		NOTE: Samples can be stored in 100% EtOH before DNA extraction, and DNA in chelation agent can be stored for two months at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Evaluation of male fertility in males produced from inbreeding crosses 
	NOTE: Diploid males produced from inbreeding crosses are often sterile.
	<ol>
		<li>Dissect internal reproductive organs in a glass dish with 400 &#181;L of PBS solution using forceps.</li>
		<li>Remove PBS and add 4% paraformaldehyde (PFA) using a micropipette.</li>
		<li>Fix the tissue by incubating in PFA for 30 min at room temperature (15 - 25 &#176;C).</li>
		<li>Wash tissue 5 times with 400 &#181;L of PBS using a micropipette.</li>
		<li>Dilute the 4&#39;,6-diamidino-2-phenylindole (DAPI) solution to 1 &#181;g/mL in PBS.</li>
		<li>Remove PBS and add approximately 300 &#181;L of this dilute DAPI staining solution to tissue.</li>
		<li>Incubate 15 min under dark condition at room temperature (15 - 25 &#176;C).</li>
		<li>Wash tissue 5 times with 400 &#181;L of PBS, and transfer tissue on center of slide glass using forceps.</li>
		<li>Mount tissue on mounting medium containing Tetramethylrhodamine (TRITC)-conjugated phalloidin.</li>
		<li>Observe samples by confocal laser scanning microscope using 20X or 63X objective lenses.</li>
		<li>Use a 405 nm excitation laser and a hybrid detector at 410-530 nm for DAPI detection.</li>
		<li>Use a 561 nm excitation laser and a hybrid detector at 565-650 nm for TRITC detection.</li>
		<li>Use a scan speed of 400 Hz (400 lines/s) at a resolution at 1024 &#215; 1024 pixels.</li>
		<li>Capture images using a software platform.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

This article demonstrates protocols that can be used to induce inbreeding crosses and evaluate the occurrence of inbreeding in the ant V. emeryi. In the experiments, genotyping of the individuals used for crosses is necessary to ensure that inbreeding crosses were successful. However, the effectiveness of these crossing tests is clearly apparent as diploid males can be produced throughout the year, while haploid males can only be produced in autumn in both the field and the laboratory6. S...

Eusocial Hymenopteran taxa, such as ants and bees, have evolved a haplodiploid sex-determination system in which individuals that are heterozygous at one or more complementary sex determination (CSD) loci become females, while those that are homo- or hemizygous become males (Figure 1A)1.
Genetic and molecular components involved in the sex determination cascade have been well studied in the honeybee, Apis mellifera, a hymenopteran model organism2,3,4. Recent comparative genomics investigations suggest that ants and honeybees share many putative homologs in the sex determination pathway, such as the initial sex determination gene, csd5. However, evidence for the functional conservation of these homologs is still lacking in ants.
To address this problem, inbreeding lines need to be developed as they are essential for genetic mapping and molecular studies. However, it is difficult to maintain and conduct experimental crosses between these organisms in the laboratory because of the complex nature of the life cycles that have evolved.
Here, we use Vollenhovia emeryi as a model to investigate the genetic and molecular basis of the sex determination system in ants6,7. The inbreeding lines of this species were developed previously for linkage mapping of quantitative trait loci (QTL) for traits related to sex determination for the first time in ants6. In addition, the molecular sex-determination cascade has been investigated7. This species has evolved an unusual reproduction system that employs both gynogenesis and androgenesis (Figure 1B)8,9. Most new queens and males are clonally produced from the maternal and paternal genomes, respectively. In addition, workers and some queens are produced sexually8. This reproduction system is particularly well suited to genetic studies because the inbreeding crosses produced using sexually produced queens and males are genetically equivalent to a classic backcross. Since sexually produced queens differ morphologically from queens produced from maternal genomes10 (Figure 1B), conducting and evaluating inbreeding crosses is greatly simplified using this method.
In this article, the methods for establishment of laboratory colonies for crossing test, application of inbreeding crosses using full-sib pairs, and evaluating the success of those crosses using genotyping of colony members and dissection of male offspring genitalia are described in V. emeryi.
Regardless of the reproduction system employed, application of inbreeding crosses is often the essential first step in any investigation of sex determination systems in the Hymenoptera. For example in Cardiocondyla obscurior, the almost complete absence of diploid males after 10 generations of full-sib mating in the laboratory demonstrates absence of CSD locus11. It is possible to predict the number of CSD loci from the ratio of males produced in inbreeding crosses6,12,13.

<table><tbody><tr><td>Plaster powder</td><td>N/A</td><td>N/A</td><td>Any brand can be used</td></tr><tr><td>Charcoal, Activated, Powder</td><td>Wako</td><td>033-02117,037-02115</td><td></td></tr><tr><td>Slide glass</td><td>N/A</td><td>N/A</td><td>Any brand can be used</td></tr><tr><td>Dry Cricket diet</td><td>N/A</td><td>N/A</td><td>Any brand can be used</td></tr><tr><td>Brown shuger&amp;nbsp;</td><td>N/A</td><td>N/A</td><td>Any brand can be used</td></tr><tr><td>Styrene Square-Shaped Case</td><td>AS ONE</td><td>Any size</td><td>Size varies by number of ants</td></tr><tr><td>Incbator</td><td></td><td></td><td>Any brand can be used</td></tr><tr><td>Aluminum block bath Dry thermo unit DTU-1B</td><td>TAITEC</td><td>0014035-000</td><td></td></tr><tr><td>1.5mL Hyper Microtube,Clear, Round bottom</td><td>WATSON</td><td>131-715CS</td><td></td></tr><tr><td>Ethanol (99.5)</td><td>Wako</td><td>054-07225</td><td></td></tr><tr><td>Stereoscopic microscope</td><td>N/A</td><td>N/A</td><td>Any brand can be used</td></tr><tr><td>Forseps</td><td>DUMONT</td><td>0108-5-PO</td><td></td></tr><tr><td>Chelex 100 sodium form</td><td>SIGMA</td><td>11139-85-8</td><td></td></tr><tr><td>Phosphate Buffer Saline (PBS) Tablets, pH7.4</td><td>TaKaRa</td><td>T9181</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Wako</td><td>162-16065</td><td></td></tr><tr><td>-Cellstain- DAPI solution</td><td>Dojindo Molecular Technologies</td><td>D523</td><td></td></tr><tr><td>ABI 3100xl Genetic Analyzer</td><td>Applied Biosystems</td><td></td><td>Directly contact the constructor formore informations.</td></tr><tr><td>Confocal laser scanning microscope Leica TCS SP8</td><td>Leica</td><td></td><td>Directly contact the constructor formore informations.</td></tr><tr><td>HC PL APO CS2 20x/0.75 IMM</td><td>Leica</td><td></td><td>Directly contact the constructor formore informations.</td></tr><tr><td>HC PL APO CS2 63x/1.20 WATER</td><td>Leica</td><td></td><td>Directly contact the constructor formore informations.</td></tr><tr><td>Leica HyDTM</td><td>Leica</td><td></td><td>Directly contact the constructor formore informations.</td></tr><tr><td>Leica Application Suite X (LAS X)</td><td>Leica</td><td></td><td>Directly contact the constructor formore informations.</td></tr></tbody></table>

induction and evaluation of inbreeding crosses using the ant, vollenhovia emeryi

The genetic and molecular components of the sex-determination cascade have been extensively studied in the honeybee, Apis mellifera, a hymenopteran model organism. However, little is known about the sex-determination mechanisms found in other non-model hymenopteran taxa, such as ants. Because of the complex nature of the life cycles that have evolved in hymenopteran species, it is difficult to maintain and conduct experimental crosses between these organisms in the laboratory. Here, we describe the methods for conducting inbreeding crosses and for evaluating the success of those crosses in ant Vollenhovia emeryi. Inducing inbreeding in the laboratory using V. emeryi, is relatively simple because of the unique biology of the species. Specifically, this species produces androgenetic males, and female reproductives exhibit wing polymorphism, which simplifies identification of the phenotypes in genetic crosses. In addition, evaluating the success of inbreeding is straightforward as males can be produced continuously by inbreeding crosses, while normal males only appear during a well-defined reproductive season in the field. Our protocol allow for using V. emeryi as a model to investigate the genetic and molecular basis of the sex determination system in ant species.

Results of microsatellite analysis using F0 and F1 generations showed that inbreeding crosses were produced successfully (Figure 4)6. As a result of inbreeding crosses, mated queens were obtained within one month of establishing the experimental crossing colonies. A quarter (27.1 &#177; 8.91% SD) of all offspring (F2) from the inbreeding crosses was male, while the remainder was female (workers and a qu...

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Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi

In this protocol, methods for conducting inbreeding crosses, and for evaluating the success of those crosses, are described for the ant Vollenhovia emeryi. These protocols are important for experiments aimed at understanding the genetic basis of sex determination systems in Hymenoptera.

Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi

The genetic and molecular components of the sex-determination cascade have been extensively studied in the honeybee, Apis mellifera, a hymenopteran model ...

induction-evaluation-inbreeding-crosses-using-ant-vollenhovia

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7.5K Views.  Utsunomiya University. In this protocol, methods for conducting inbreeding crosses, and for evaluating the success of those crosses, are described for the ant Vollenhovia emeryi. These protocols are important for experiments aimed at understanding the genetic basis of sex determination systems in Hymenoptera.

Video: Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi

Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses

Setaria viridis is an emerging model system for C4 grasses. It is closely related to the bioenergy feed stock switchgrass and the grain crop foxtail millet. Recently, the 510 Mb genome of foxtail millet, S. italica, has been sequenced 1,2 and a 25x coverage genome sequence of the weedy relative S. viridis is in progress. S. viridis has a number of characteristics that make it a potentially excellent model genetic system including a rapid generation time, small stature, simple growth requirements, prolific seed production 3 and developed systems for both transient and stable transformation 4. However, the genetics of S. viridis is largely unexplored, in part, due to the lack of detailed methods for performing crosses. To date, no standard protocol has been adopted that will permit rapid production of seeds from controlled crosses.
The protocol presented here is optimized for performing genetic crosses in S. viridis, accession A10.1. We have employed a simple heat treatment with warm water for emasculation after pruning the panicle to retain 20-30 florets and labeling of flowers to eliminate seeds resulting from newly developed flowers after emasculation. After testing a series of heat treatments at permissive temperatures and varying the duration of dipping, we have established an optimum temperature and time range of 48 &deg;C for 3-6 min. By using this method, a minimum of 15 crosses can be performed by a single worker per day and an average of 3-5 outcross progeny per panicle can be recovered. Therefore, an average of 45-75 outcross progeny can be produced by one person in a single day. Broad implementation of this technique will facilitate the development of recombinant inbred line populations of S. viridis X S. viridis or S. viridis X S. italica, mapping mutations through bulk segregant analysis and creating higher order mutants for genetic analysis.

Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses

We have developed a methodology for performing crosses in Setaria viridis (S. viridis). The method involves pruning the panicle prior to a hot water treatment to kill viable pollen. Crosses are performed following a well-controlled growth regime and typically result in the recovery of 1 to 7 cross-pollinated seed/s per panicle.

Setaria viridis is an emerging model system for C4 grasses. It is closely related to the bioenergy feed stock switchgrass and the grain crop foxtail ...

Environment

Protocol for Assessing the Relative Effects of Environment and Genetics on Antler and Body Growth for a Long-lived Cervid

Cervid phenotype can be placed into one of two categories: efficiency, which promotes survival over extravagant morphometric growth, and luxury, which promotes growth of large weaponry and body size. Populations of the same species display each phenotype depending on environmental conditions. Although antler and body size of male white-tailed deer (Odocoileus virginianus) varies by physiographic region in Mississippi, USA and is strongly correlated with regional variation in nutritional quality, the effects of population-level genetics from native stocks and previous re-stocking efforts cannot be disregarded. This protocol describes how we designed a controlled study, where other factors that influence phenotype, such as age and nutrition, are controlled. We brought wild-caught pregnant females and six-month-old fawns from three distinct physiographic regions in Mississippi, USA to the Mississippi State University Rusty Dawkins Memorial Deer Unit. Deer from the same region were bred to produce a second generation of offspring, allowing us to assess generational responses and maternal effects. All deer ate the same high-quality (20% crude protein deer pellet) diet ad libitum. We uniquely marked each neonate and recorded body mass, hind foot, and total body length. Each subsequent fall, we sedated individuals via remote injection and sampled the same morphometrics plus antlers of adults. We found that all morphometrics increased in size from first to second generation, with full compensation of antler size (regional variation no longer present) and partial compensation of body mass (some evidence of regional variation) evident in the second generation. Second generation males that originated from our poorest quality soil region displayed about a 40% increase in antler size and about a 25% increase in body mass when compared to their wild harvested counterparts. Our results suggest phenotypic variation of wild male white-tailed deer in Mississippi are more related to differences in nutritional quality than population-level genetics.

Phenotypic differences among cervid populations may be related to population-level genetics or nutrition; discerning which is difficult in the wild. This protocol describes how we designed a controlled study where nutritional variation was eliminated. We found that phenotypic variation of male white-tailed deer was more limited by nutrition than genetics.

Cervid phenotype can be placed into one of two categories: efficiency, which promotes survival over extravagant morphometric growth, and luxury, which ...

In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors

Although solitary bees provide crucial pollination services for wild and managed crops, this species-rich group has been largely overlooked in pesticide regulation studies. The risk of exposure to fungicide residues is likely to be especially high if the spray occurs on, or near host plants while the bees are collecting pollen to provision their nests. For species of Osmia that consume pollen from a select group of plants (oligolecty), the inability to use pollen from non-host plants can increase their risk factor for fungicide-related toxicity. This manuscript describes protocols used to successfully rear oligolectic mason bees, Osmia ribifloris sensu lato, from egg to prepupal stage within cell culture plates under standardized laboratory conditions. The in vitro-reared bees are subsequently used to investigate the effects of fungicide exposure and pollen source on bee fitness. Based on a 2 &#215; 2 fully crossed factorial design, the experiment examines the main and interactive effects of fungicide exposure and pollen source on larval fitness, quantified by prepupal biomass, larval developmental time, and survivorship. A major advantage of this technique is that using in vitro-reared bees reduces natural background variability and allows the simultaneous manipulation of multiple experimental parameters. The described protocol presents a versatile tool for hypotheses testing involving the suite of factors affecting bee health. For conservation efforts to be met with significant, lasting success, such insights into the complex interplay of physiological and environmental factors driving bee declines will prove to be critical.

In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors

Fungicide sprays on flowering plants may expose solitary bees to high concentrations of pollen-borne fungicide residues. Using laboratory-based experiments involving in vitro-reared bee larvae, this study investigates the interactive effects of consuming fungicide-treated pollen derived from host and non-host plants.

Although solitary bees provide crucial pollination services for wild and managed crops, this species-rich group has been largely overlooked in pesticide ...

Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator

The unique traits of eusocial insects, such as social behavior and reproductive division of labor, are controlled by their genetic system. To address how genes regulate social traits, we have developed mutant ants via delivery of CRISPR complex into young embryos during their syncytial stage. Here, we provide a protocol of CRISPR-mediated mutagenesis in Harpegnathos saltator, a ponerine ant species that displays striking phenotypic plasticity. H. saltator ants are readily reared in a laboratory setting. Embryos are collected for microinjection with Cas9 proteins and in vitro synthesized small guide RNAs (sgRNAs) using home-made quartz needles. Post-injection embryos are reared outside the colony. Following emergence of the first larva, all embryos and larvae are transported to a nest box with a few nursing workers for further development. This protocol is suitable for inducing mutagenesis for analysis of caste-specific physiology and social behavior in ants, but may also be applied to a broader spectrum of hymenopterans and other insects.

Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator

Many characteristics of insect eusociality rely on within-colony communication and division of labor. Genetic manipulation of key regulatory genes in ant embryos via microinjection and CRISPR-mediated mutagenesis provides insights into the nature of altruistic behavior in eusocial insects.

The unique traits of eusocial insects, such as social behavior and reproductive division of labor, are controlled by their genetic system. To address how ...

Mass-Rearing and Molecular Studies in Tortricidae Pest Insects

Tortricidae (Lepidoptera), commonly known as tortrix or leafroller moths, comprises many agricultural and forestry pests, which cause serious agricultural losses. To understand the biology of such pest moths, fundamental techniques have been in high demand. Here, methods for mass-rearing, observations, and molecular studies are developed using two tea tortrix, Homona magnanima and Adoxophyes honmai (Lepidoptera: Tortricidae). Insects were mass-reared with sliced artificial diet and maintained by inbreeding for over 100 generations by considering their biological characteristics. Insects have various sex dimorphisms; hence it is difficult to distinguish the sex during the developing stages, which have prevented subsequent assays. The present work highlighted that the sex of tortricids larvae could be determined by observing testes or lactic-acetic orcein staining to visualize the female-specific W chromosome. Moreover, using the sex determination methods, the present study enabled nucleic acid extractions from sex determined embryos and application toward high throughput sequencing. These tips are applicable for other pest insects and will facilitate further morphological and genetic studies.

The present protocol describes the rearing method of tortricid pest insects in the laboratories. The procedures to distinguish insects' sex and extract nucleic acids for high throughput sequencing are established using two tortricid pests.

Tortricidae (Lepidoptera), commonly known as tortrix or leafroller moths, comprises many agricultural and forestry pests, which cause serious agricultural ...

Measuring Embryonic Viability and Brood Size in Caenorhabditis elegans

Caenorhabditis elegans is an excellent model organism for the study of meiosis, fertilization, and embryonic development. C. elegans exist as self-fertilizing hermaphrodites, which produce large broods of progeny-when males are present, they can produce even larger broods of cross progeny. Errors in meiosis, fertilization, and embryogenesis can be rapidly assessed as phenotypes of sterility, reduced fertility, or embryonic lethality. This article describes a method to determine embryonic viability and brood size in C. elegans. We demonstrate how to set up this assay by picking a worm onto an individual Modified Youngren's, Only Bacto-peptone (MYOB) plate, establish the appropriate timeframe to count viable progeny and non-viable embryos, and explain how to accurately count live worm specimens. This technique can be used to determine viability in self-fertilizing hermaphrodites as well as cross-fertilization by mating pairs. These relatively simple experiments are easily adoptable for new researchers, such as undergraduate students and first-year graduate students.

Measuring Embryonic Viability and Brood Size in Caenorhabditis elegans

Here, we present a general method to determine the embryonic viability and total number of embryos produced (brood) using the model organism C. elegans.

Caenorhabditis elegans is an excellent model organism for the study of meiosis, fertilization, and embryonic development. C. elegans exist as ...

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or complete loss of viability at different stages of development. Our lab has developed a method to assess viability in Drosophila from embryo to fully mature adult. The method relies on quantifying the number of progeny present at different stages during development, starting with hatched embryos. After embryos have been quantified, additional stages are counted, including L1/L2, pupae, and mature adults. After all stages have been examined, a statistical analysis such as the chi-square test is used to determine if there is a significant difference between the starting number of progeny (hatched embryos) and later stages culminating in the observed number of adults, thus rejecting or accepting the null hypothesis (that the number of hatched embryos will be equal to the number of larvae, pupae, and adults recorded throughout the stages of development). The primary advantage of this assay is its simplicity and accuracy, as it does not require an embryo rinse to transfer them to the food vial, avoiding losses from technical errors. Although the protocol described here does not directly examine L2/L3 larvae, additional steps can be added to account for these. Comparing the number of hatched embryos, L1, pupae, and adults can help determine if viability was compromised during the L2/L3 stages for further studies (the use of colored food helps with visual identification of larvae). Overall, this method can help Drosophila researchers and educators determine when viability is compromised during the fly life cycle. Routine assessment of stocks using this assay can prevent accumulation of secondary mutations that may affect the phenotype of the originally isolated mutant, especially if the original mutations affect fitness. For this reason, our lab maintains multiple copies of each of our Dm ime4 alleles and routinely checks the purity of each stock with this method in addition to other molecular analyses.&#160;

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

This protocol is designed to assess the viability of Drosophila at every developmental stage, from embryo to adult. The method can be used to determine and compare the viability of different genotypes or growth conditions.

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or ...

Developmental Biology

Determination of Self- and Inter-(in)compatibility Relationships in Apricot Combining Hand-Pollination, Microscopy and Genetic Analyses

Self-incompatibility in Rosaceae is determined by a Gametophytic Self-Incompatibility System (GSI) that is mainly controlled by the multiallelic locus S. In apricot, the determination of self- and inter-(in)compatibility relationships is increasingly important, since the release of an important number of new cultivars has resulted in the increase of cultivars with unknown pollination requirements. Here, we describe a methodology that combines the determination of self-(in)compatibility by hand-pollinations and microscopy with the identification of the S-genotype by PCR analysis. For self-(in)compatibility determination, flowers at balloon stage from each cultivar were collected in the field, hand-pollinated in the laboratory, fixed, and stained with aniline blue for the observation of pollen tube behavior under the fluorescence microscopy. For the establishment of incompatibility relationships between cultivars, DNA from each cultivar was extracted from young leaves and S-alleles were identified by PCR. This approach allows establishing incompatibility groups and elucidate incompatibility relationships between cultivars, which provides a valuable information to choose suitable pollinizers in the design of new orchards and to select appropriate parents in breeding programs.

Determination of Self- and Inter-incompatibility Relationships in Apricot Combining Hand-Pollination, Microscopy and Genetic Analyses

We present a methodology to establish the pollination requirements of apricot (Prunus armeniaca L.) cultivars combining the determination of self-(in)compatibility by fluorescence microscopy with the identification of the S-genotype by PCR analysis.

Self-incompatibility in Rosaceae is determined by a Gametophytic Self-Incompatibility System (GSI) that is mainly controlled by the multiallelic locus S. ...

Assessing Differences in Sperm Competitive Ability in Drosophila

Competition among conspecific males for fertilizing the ova is one of the mechanisms of sexual selection, i.e. selection that operates on maximizing the number of successful mating events rather than on maximizing survival and viability 1. Sperm competition represents the competition between males after copulating with the same female 2, in which their sperm are coincidental in time and space. This phenomenon has been reported in multiple species of plants and animals 3. For example, wild-caught D. melanogaster females usually contain sperm from 2-3 males 4. The sperm are stored in specialized organs with limited storage capacity, which might lead to the direct competition of the sperm from different males 2,5.
Comparing sperm competitive ability of different males of interest (experimental male types) has been performed through controlled double-mating experiments in the laboratory 6,7. Briefly, a single female is exposed to two different males consecutively, one experimental male and one cross-mating reference male. The same mating scheme is then followed using other experimental male types thus facilitating the indirect comparison of the competitive ability of their sperm through a common reference. The fraction of individuals fathered by the experimental and reference males is identified using markers, which allows one to estimate sperm competitive ability using simple mathematical expressions 7,8. In addition, sperm competitive ability can be estimated in two different scenarios depending on whether the experimental male is second or first to mate (offense and defense assay, respectively) 9, which is assumed to be reflective of different competence attributes.
Here, we describe an approach that helps to interrogate the role of different genetic factors that putatively underlie the phenomenon of sperm competitive ability in D. melanogaster.

Assessing Differences in Sperm Competitive Ability in Drosophila

Differential sperm competitive ability among Drosophila males with distinct genotypes can be ascertained through double-mating experiments. Each of these experiments involves one of the males of interest and a reference male. Readily identifiable markers in the progeny allow inference of the fraction of individuals fathered by each male.

Competition among conspecific males for fertilizing the ova is one of the mechanisms of sexual selection, i.e. selection that operates on maximizing the ...

Biology

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. To improve the mating efficiency of B. Xylophilus and to increase the chances of mating observation, virgin adults were cultured and used for the investigation. Eggs were obtained by keeping the nematodes in water and allowing the females to lay eggs for 10 min. The second-stage juveniles (J2) were synchronized by incubating the eggs for 24 h at 25 &#176;C in the dark, and the early J4 were obtained by culturing the J2 with grey mold, Botrytis cinerea, for another 52 h. At this time point, most J4 nematodes could be clearly distinguished as being male or female using their genital morphology. The male and female J4 were collected and cultured separately in two different Petri dishes for 24 h to get virgin adult nematodes. A virgin male and a virgin female were paired in a drop of water in the well of a concave slide. The mating behavior was filmed with a video recorder under a stereomicroscope. The whole period of the mating process was 82.8 &#177;3.91 min (mean &#177;SE) and could be divided into 4 different phases: searching, contacting, copulating, and lingering. The mean minutes of duration were 21.8 &#177; 2.0, 28.0 &#177; 1.9, 25.8 &#177; 0.7 and 7.2 &#177; 0.5, respectively. Eleven sub-behaviors were described: cruising, approaching, encountering, touching, hooping, locating, attaching, ejaculating, separating, quiescence, and roaming. Interestingly, obvious intra-sexual competition was observed when one female was grouped with 3 males or one male with 3 females. This protocol is useful and valuable, not only in investigating the mating behavior of B. xylophilus, but also in acting as a reference for ethological studies of other nematodes.

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A protocol for investigating the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, is presented. Behavioral features of B. xylophilus are described in the mating process.

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. ...

Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi

Summary

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Chapters in this video

Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses

Protocol for Assessing the Relative Effects of Environment and Genetics on Antler and Body Growth for a Long-lived Cervid

In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors

Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator

Mass-Rearing and Molecular Studies in Tortricidae Pest Insects

Measuring Embryonic Viability and Brood Size in Caenorhabditis elegans

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

Determination of Self- and Inter-(in)compatibility Relationships in Apricot Combining Hand-Pollination, Microscopy and Genetic Analyses

Assessing Differences in Sperm Competitive Ability in Drosophila

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus