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. Sib-mated queens start to produce male offspring immediately after crossing. No morphological phenotypic differences were observed between diploid and haploid males in V. emeryi6,7. However, diploid male V. emeryi almost invariably fail to develop testes. In the absence of genetic markers for testing the genetic relatedness of pairs used for crossing tests, the reproductive potential of male offspring can be used to infer whether inbreeding has occurred. However, it should be noted that males produced in inbreeding crosses are not always sterile in other hymenopteran species15,16,17.
The first critical step in the success of the protocol is the maintenance of well-fed colonies, as feeding them after field collection will increase the likelihood of obtaining sufficient numbers of reproductives for crosses. In V. emeryi, a positive correlation has been reported between nutrition and the production of long-winged queens, which are the queens used for the inbreeding crosses10. In social insects, small colonies, or colonies in poor health, tend not to produce new reproductives18. It is therefore important to collect mature colonies from the field and to provide them with adequate amounts of nutritious food for experiments using new reproductives.
The second critical step is to keep workers, reproductive and few larvae or pupae together during the crossing tests and to maintain the experimental crossing colony at the same state as that of a normal colony until crossing is completed (for a week to a month). It is difficult to maintain colonies that are to be used for crossing tests without workers for more than 3 days because males are unable to feed themselves and must be fed by workers. Under such unnatural conditions, the success rate of inbreeding crosses was extremely low6.
There are two limitations regarding the application of these protocols to other ant species. First, the cues for inducing crosses are species specific. It is relatively easy to induce laboratory crosses in V. emeryi since intra-colony mating without flight occurs in nature. However, many ant species have evolved mating rituals that involve nuptial flights during which new queens and males mate during or after flight19. It is therefore important to elucidate the triggers that induce crossing in each species in a laboratory setting. For example, in the parasitic ant Acromyrmex ameliae, the main stimulus for triggering nuptial flights appears to be light20. The second limitation is, in some cases, the diploid males produced by inbreeding crosses cannot be collected as they are inviable or killed by workers because they do not work and/or have no or low reproductive potential, and are thus a major cost to the colony of the objective species21,22,23. Fortunately, diploid V. emeryi males are not killed by workers and they live until they die naturally, which suggests that they are not frequently encountered in nature and a strategy to exclude diploid males from colonies has not evolved in this species.
Compared to other ant species that employ arrhenotokous parthenogenesis to reproduce (Figure 1A), we can assume that there are certain advantages to producing experimental inbreeding crosses using V. emeryi by androgenesis as the inbreeding crosses are genetically equivalent to a classic backcross. Indeed, the system has enabled us to design experiments to investigate the sex-determination genes, molecular mechanisms, and perform functional studies in this species6,7.
In summary, methods to conduct inbreeding crosses and to evaluate the success of the resulting crosses have been described in V. emeryi. These protocols are essential for experiments directed at understanding the genetic and molecular basis of sex determination systems in the Hymenoptera.

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 queen)6. QTL mapping using the offspring from inbreeding crosses showed that males produced by inbreeding crosses were diploid and homozygous at two CSD loci (CSD1 and CSD2 in Figure 5), while females (workers) produced from inbreeding crosses were diploid and heterozygous at least at one CSD locus6.
Dissection of haploid males revealed testes and sperm, as expected (Figure&#160;6A-6C). However, in diploid males, sperms were never observed, suggesting that males produced in inbreeding crosses are sterile in V. emeryi6. In addition, testes of diploid males failed to develop&#160;(Figure&#160;6D-6E).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58521/58521fig1.jpg" /> 
Figure 1. Typical reproductive system in (A) Hymenoptera and the atypical reproductive system involving androgenesis and gynogenesis in (B) V. emeryi. Typically, females (workers and queens) develop from fertilized diploid eggs, and males develop from unfertilized haploid eggs containing half of the maternal genome (A). In V. emeryi, sterile workers and a few long-winged queens (LWQ) develop from fertilized diploid eggs, while short-winged queens (SWQ) develop with nearly complete maternal genomes from unfertilized diploid eggs (gynogenesis). Males never inherit maternal genomes but are clones of their fathers (androgenesis) (B). This figure has been modified from [Miyakawa et al. 2018]7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58521/58521fig1large.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/58521/58521fig2.jpg" /> 
Figure 2. Experimental set up of V. emeryi colonies. After field collection, colonies are transferred into an artificial plaster nest and kept in the laboratory. A large plaster nest (left) is prepared for maintain collected colonies, whereas a smaller plaster nest (right) is prepared for experimental inbreeding crosses. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58521/58521fig2large.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/58521/58521fig3.jpg" /> 
Figure 3. New V. emeryi reproductives emerge during the reproductive season. Mature and well-fedcolonies tend to produce long-winged queens (LWQ) with the parental genome in addition to short-winged queens (SWQ) which bear only the maternal genome (Figure 1B). Photo courtesy of Mr. Taku Shimada. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58521/58521fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58521/58521fig4.jpg" /> 
Figure 4. Design of inbreeding crosses and microsatellite genotypes of F0 and F1 generations. Using 11 microsatellite markers developed in previous studies6,8,9, females and males of the parental generation (F0) showed different genotypes. The genotypes of females and males used for experimental crosses (F1) inherited the parental and paternal genotypes, respectively, indicating that females were crossed successfully with their brothers, with which the females shared half their genomes. Numbers indicate lengths of PCR products at microsatellite locus L-5, which is one of the markers used for genotyping6,8,9,10. This figure has been illustrated according to the data from [Miyakawa and Mikheyev 2015]6. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58521/58521fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58521/58521fig5.jpg" /> 
Figure 5. Allele patterns of two CSD loci (CSD1 and CSD2) in offspring produced by inbreeding crosses. Proportion of diploid males (about 25%) and QTL mapping using offspring produced by sib-mated queens suggest the existence of two CSD loci in V. emeryi. Females are heterozygous in at least one of the two CSD loci whereas males are homozygous at all loci. Genotypes are represented by letters of the alphabet. This figure has been modified from [Miyakawa et al. 2018]7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58521/58521fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58521/58521fig6.jpg" /> 
Figure 6. Male internal reproductive organs of androgenetic haploid and diploid males in V. emeryi. Morphologies of testes and other internal reproductive organs dissected out from the androgenetic haploid males (A). Sperm (fibrous tissue) could be seen in testes of haploid males (B and C). Blue color marks nuclei stained by DAPI, and red color marks F-actin stained by Tetramethylrhodamine (TRITC)-conjugated phalloidin in B, C, and E. (a) accessory glands; (t) testes; (v) vas deferens; (g) external genitalia. Morphologies of internal reproductive organs dissected out from the diploid males (D). Testes and sperm of diploid males were never observed (D and E, N &#62;&#62; 30). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58521/58521fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi at JoVE.com

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

Nanyang Technological University

Research

JoVE Journal

Genetics

7.5K Views.  Utsunomiya University. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in de moleculaire biologie en genetische sferen van Hymenoptera, zoals geslachtsbepalingssystemen. Het belangrijkste voordeel van deze techniek is dat we gemakkelijk inteeltlijnen kunnen opwekken over de mier Vollenhovia Emery, die essentieel zijn voor genetische mapping en de moleculaire status. Dus, deze missie kan inzicht geven in het geslacht bepaling systeem van een specifieke mier en het kan ook worden toegepast op ande...

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

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 ...

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors remains poorly understood. In Drosophila melanogaster, environmental cues, such as food availability, affect mating activity offering a tractable system to investigate the mechanisms modulating sexual behavior. In D. melanogaster, environmental cues are often sensed via the chemosensory gustatory and olfactory systems. Here, we present a method to test the effect of environmental chemical cues on mating behavior. The assay consists of a small mating arena containing food medium and a mating couple. The mating frequency for each couple is continuously monitored for 24 h. Here we present the applicability of this assay to test environmental compounds from an external source through a pressurized air system as well as manipulation of the environmental components directly in the mating arena. The use of a pressurized air system is especially useful to test the effect of very volatile compounds, while manipulating components directly in the mating arena can be of value to ascertain a compound's presence. This assay can be adapted to answer questions about the influence of genetic and environmental cues on mating behavior and fecundity as well as other male and female reproductive behaviors.

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

We demonstrate an assay to analyze the environmental and genetic cues that influence mating behavior in the fruit fly Drosophila melanogaster.

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors ...

Behavior

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted reproduction also allows for the production of embryos from high performers without interrupting their sports career and contributes to an increase in the number of foals from mares of high genetic value. The present manuscript describes the procedures used for collecting immature and mature oocytes from horse ovaries using ovum pick-up (OPU). These oocytes were then used to investigate the incidence of aneuploidy by adapting a protocol previously developed in mice. Specifically, the chromosomes and the centromeres of metaphase II (MII) oocytes were fluorescently labeled and counted on sequential focal plans after confocal laser microscope scanning. This analysis revealed a higher incidence in the aneuploidy rate when immature oocytes were collected from the follicles and matured in vitro compared to in vivo. Immunostaining for tubulin and the acetylated form of histone four at specific lysine residues also revealed differences in the morphology of the meiotic spindle and in the global pattern of histone acetylation. Finally, the expression of mRNAs coding for histone deacetylases (HDACs) and acetyl-transferases (HATs) was investigated by reverse transcription and quantitative-PCR (q-PCR). No differences in the relative expression of transcripts were observed between in vitro and in vivo matured oocytes. In agreement with a general silencing of the transcriptional activity during oocyte maturation, the analysis of the total transcript amount can only reveal mRNA stability or degradation. Therefore, these findings indicate that other translational and post-translational regulations might be affected.
Overall, the present study describes an experimental approach to morphologically and biochemically characterize the horse oocyte, a cell type that is extremely challenging to study due to low sample availability. However, it can expand our knowledge on the reproductive biology and infertility in monovulatory species.

This manuscript describes an experimental approach to morphologically and biochemically characterize horse oocytes. Specifically, the present work illustrates how to collect immature and mature horse oocytes by ultrasound-guided ovum pick-up (OPU) and how to investigate chromosome segregation, spindle morphology, global histone acetylation, and mRNA expression.

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted ...

Developmental Biology

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 ...

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. ...

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2 When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3 Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5 by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.6-8

A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

Biology

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 ...

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

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