Dedicated to Mario Autuori (in memoriam) and Walter Hugo de Andrade Cunha who greatly contributed to the leaf-cutting ant studies. We acknowledge the support of S&#227;o Paulo State University and the Institute of Biosciences. This study was in part financed by the Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior-Brasil (CAPES) - Finance Code 001, Conselho Nacional de Desenvolvimento Cient&#237;fico e Tecnol&#243;gico (CNPq), Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado de S&#227;o Paulo (FAPESP), and Funda&#231;&#227;o para o Desenvolvimento da UNESP (Fundunesp).

The authors have no conflicts of interest to disclose.

The protocol described here to maintain leaf-cutting ant colonies has been developed and applied for over three decades in an assertive and replicable way. It allowed the development of research that would be limited by field conditions. Thereby, healthy ants and colonies became available for research in several areas such as comparative morphology, toxicology51,52, histology53, and microbiology54,<sup cl...

Ants compose a diverse group of individuals that exert an influence on most terrestrial environments1. They act as efficient dispersers2,3,4, predators5 and ecosystem engineers6,7,8,9,10, highlighting their importance and ecological success on natural ecosystems. All ant species are classified as eusocial insects; however, their social organization varies greatly among different species groups, i.e., labor division systems, functional groups, communication among individuals, forage organization, colony foundation, and reproduction process11. As a highly diversified group, they resort to several food resources and specialized feeding behaviors. As a matter of fact, agriculture was not only a huge step for human civilization, but also for ant species. Approximately 55 to 65 Ma ago12, attine ants began to culture fungi and incorporate them into an almost exclusive diet. They became so specialized that they developed strict, dependent, and obligatory interactions classified as symbiosis, where one individual does not survive without the other.
Lower fungus-growing ants collect and process dead organic matter, such as fragments of rotting leaf, to grow their mutualistic fungus; while higher fungus-growing ants harvest fresh plant material, composing one of the most successful symbiotic natural systems13. This highly specialized agriculture technique allowed them to seize a new niche. The higher attine ants comprise the leaf-cutting ants, a monophyletic group that arouse between 19 Ma (15-24 Ma) and 18 Ma (14-22 Ma)14,15,16 consisting of four valid genera: Atta Fabricius, Acromyrmex Mayr, Amoimyrmex Cristiano, and Pseudoatta Gallardo. The leaf-cutter agriculture system performed by the leaf-cutting ants, evolved from derived agriculture systems17. Most of these species exclusively exploit the mutualistic fungus species Leucoagaricus gongylophorus Singer18 (also called Leucocoprinus gongylophorus Heim19), marking a significant evolutionary transition11. The fungal cultivars are transmitted vertically, from original nests to offsprings, suggesting that they are clonally propagated20.
Remarkably, Atta societies developed a complex organizational structure of enormous importance in their environment and of great interest to myrmecologists. Their population can be composed of millions of individuals, most of them sterile female workers that display an accentuated polymorphism, i.e., distinct size and anatomical morphology. The population is distinguished by castes according to age, physiological state, morphological type, behaviors, and specialized activities in the colony21. Workers can be discriminated into gardeners and nurses, within-nest generalists, foragers and excavators, and defenders or soldiers21. This organization allows the performance of tasks in cooperation and a self-organizing system that can produce highly structured collective behaviors, allowing them to respond efficiently to environmental disturbances22.
The role of population renewal is played by a single queen (i.e., monogynous), for as long as she lives, constituting the permanent reproductive caste22. Atta queens are known to live for more than 20 years, laying eggs throughout their lifespan23. As the queen is irreplaceable, its endurance is crucial for the survival of the colony13,20,23,24. Yet, thousands of winged reproductive females and males can be found in the nest during breeding seasons, but none stays in the original nest, forming a temporary caste22. In Atta sexdens colonies, nearly 3,000 reproductive females and 14,000 reproductive males are produced25. It occurs when a colony reaches sexual maturity, approximately 38 months from its implementation, and is repeated annually ever since until it is extinguished23,25. New Atta colonies are established through haplometrosis, where one queen commences a new nest.
When environmental conditions are favorable, the reproducers leave the underground nest to begin the nuptial flight. The period of its occurrence differs by region, ranging along the year throughout the brazilian territory depending on the species. However, the event seems to be preceded by rainfalls and humidity elevation26, which can be related to excavation facilitation due to soil moisture22. Frequently, 1-5 weeks before the nuptial flight, nest entrances and channels are widened to facilitate the reproductive individuals to depart. Before leaving their mother colonies, the winged females collect and store, in an infrabuccal cavity, a portion of the mutualistic fungus20,27. Multiple copulations are performed mid-flight, and it is calculated that one queen can be inseminated by three to eight males (i.e., polyandry) in some species28, ensuring genetic variability29. Afterward, the queens proceed to the soil, giving preference to locations with no or few vegetation25, where they remove their wings and excavate their first nest chamber. This is the only period where queens can be seen outside the nest. Although individuals of the temporary caste were seen in artificial nests, it is unknown whether any successful copulation (i.e., nuptial flight) was performed in laboratory conditions24.
The initial nest construction corresponds to the most crucial period of the colony, which can last from 6 h to 8 h23,25. At this moment, the queen cloisters herself in the initial chamber, and in a matter of days, oviposition begins. The first eggs are fed to the mycelial that the queen regurgitates, marking the start of the colony&#39;s fungus garden. The first larvae appear in approximately 25 days22, and nearly at the end of the first month, the colony consists of a mat of proliferating fungus, where immatures (eggs, larvae, and pupae) are nested, and the queen, who raises her initial offspring in isolation23. Eggs are also the food resource of the first larvae and highly consumed by the queen13. Additionally, the queen sustains herself with fat-body reserves and catabolizing wing muscles that are no longer of use13. The initial fungus culture is not consumed as the colony survival depends on its development, and during this period, the queen fertilizes it with fecal fluid13. Days after emerging, the first workers open the nest entrance and begin a foraging activity in the immediate area of the nest13. They incorporate the material collected as the substrate of the fungus garden, which is now serving as food for the workers13,22. Before being added to the fungus culture, the plant material carried in by the workers is cut into tiny pieces and moistened with fecal liquid13. The ants manipulate fungus inoculum to increase and control its growth, which will serve for partitioning big soil excavated chambers, specialized in conditioning the garden13,22,25.
About 6 months after the nuptial flight, A. sexdens nests contain a fungus chamber and a few channels. The great specialization in the construction of leaf-cutting ant nests works as a defense mechanism against natural enemies and unfavorable environmental factors22. Leaf-cutting ants are known to fragment the fungus garden and transpose it to chambers with high humidity when chambers start to dry out13. Thus, despite the excavation of the nest having a considerable energy cost, the energy invested is reversed in benefits for the colony itself22. With a few exceptions, Atta species also make specialized chambers for the colony&#39;s waste, made mostly of depleted fungus substrate and bodies of dead ants, isolating it from the rest of the nest, and establishing an important social immunity strategy30. In addition, a distinct group of workers manipulate the refuse directly, to avoid contamination of other individuals. Workers constantly forage to nurture the fungus, which is the main nutritional resource of the colony. However, they can feed on plant sap as well while cutting fragments. Plant material is carefully selected for the fungus garden maintenance and influenced by many factors such as leaf traits and properties of the ecosystem13.
The foraging strategy of leaf-cutting ants to obtain fresh material is highly complex, and combined with the high harvest demand of established colonies, result in considerable economic loss to agricultural producers and jeopardizes forest restoration areas22,31. Therefore, these ants can be categorized as pests in most areas where they may be encountered, ranging from southern United States to north-eastern Argentina11,13,22,32. The extinguishing of problematic colonies is challenging due to the series of adaptations inherent in the biology of these insects (i.e., social organization, foraging, fungus-cultivation, hygiene, and complex nest structures)33. Hence, the population control strategies are distinct from those generally applied to other insect pests, and mainly resort to attractive contaminated bait offerings33,34. However, as these ants can reject harmful substances for both the fungus and the colony individuals, and compromise cultivated fields33, new natural compounds and alternatives of control are constantly being tested33,35,36. As experiment results can hardly be monitored on field-tested colonies, preliminary essays are conducted in a controlled environment.
Thus, experimental protocols must be adapted to groups of interest considering the heterogeneous lifestyle of ants, supporting studies on a species level, and accounting for colonies as operational units, where one ant is an element of a complex superorganism11. The reports gathered until now concerning the Atta genus made it attainable to successfully collect and maintain colonies in laboratory conditions and acknowledge their basic needs and general functioning. Based on their natural processes such as reproduction, colony founding, and feeding behaviors, a routine of practices has been developed that permits the long-term establishment of colonies in different types of nests. Here, a procedural protocol to maintain leaf-cutting ants in the laboratory is described and highlights possible general research with distinct experimentation purposes and science outreach.

<table><tbody><tr><td>Entomologic forceps</td><td>N/A</td><td>N/A</td><td>N/A</td></tr><tr><td>Glass tank</td><td>N/A</td><td>N/A</td><td>Tempered glass, custom made</td></tr><tr><td>Hose</td><td>N/A</td><td>N/A</td><td>Transparent, PVC 1/2 Inch x 2,0 mm</td></tr><tr><td>Latex gloves</td><td>Descarpack</td><td>550301</td><td>N/A</td></tr><tr><td>Nitrile gloves</td><td>Descarpack</td><td>433301</td><td>N/A</td></tr><tr><td>Open arena</td><td>N/A</td><td>N/A</td><td>Polypropylene crate</td></tr><tr><td>Plaster pouder</td><td>N/A</td><td>N/A</td><td>Plaster pouder used in construction, must be absorbant</td></tr><tr><td>Plastic Containers for collection</td><td>Prafesta</td><td>Natural C&amp;oacute;d.: 8231/Natural C&amp;oacute;d.: 8262</td><td>Lidded, transparent , polypropylene</td></tr><tr><td>Plastic containers for nests</td><td>Prafesta</td><td>Discontinued</td><td>Polystyrene, hermetic</td></tr><tr><td>Teflon</td><td>Dupont</td><td>N/A</td><td>Polytetrafluoroethylene liquid (PTFE Dispertion 30)</td></tr></tbody></table>

collection and long-term maintenance of leaf-cutting ants (atta) in laboratory conditions

Ants are one of the most biodiverse groups of animals on the planet and inhabit different environments. The maintenance of ant colonies in controlled environments enables an enriched comprehension of their biology that can contribute to applied research. This practice is usually employed in population control studies of species that cause economic loss, such as Atta ants. To cultivate their mutualistic fungus, these leaf-cutting ants collect leaves and for this are considered agricultural pests widely distributed throughout the American continent. They are highly socially organized and inhabit elaborated underground nests composed of a variety of chambers. Their maintenance in a controlled environment depends on a daily routine of several procedures and frequent care that are described here. It starts with the collection of queens during the reproductive season (i.e., nuptial flight), which are then individually transferred to plastic containers. Due to the high mortality rate of queens, a second collection can be carried out about 6 months after the nuptial flight, when incipient nests with developed fungus wad are excavated, hand-picked, and placed in plastic containers. In the laboratory, leaves are daily provided to established colonies, and ant-produced waste is weekly removed along with remaining dry plant material. As the fungus garden keeps growing, colonies are transferred to different types of containers according to the experimental purpose. Leaf-cutting ant colonies are placed in interconnected containers, representing the organizational system with functional chambers built by those insects in nature. This setup is ideal to monitor factors such as waste amount, fungus garden health, and the behavior of workers and queen. Facilitated data collection and more detailed observations are considered the greatest advantage of keeping ant colonies in controlled conditions.

A flowchart depicting the process of ant collection is shown in Figure 6. Here, some results obtained employing the protocol of collection, maintenance, and nest setups described above are shown.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64154/64154fig6v2.jpg" /> 
Figure 6: Flowchart for collection of leaf-cutting ants&#39; colonies. Following the protoc...

Watch this Scientific Journal Video about Collection and Long-Term Maintenance of Leaf-Cutting Ants Atta in Laboratory Conditions at JoVE.com

Collection and Long-Term Maintenance of Leaf-Cutting Ants Atta in Laboratory Conditions

Here, a protocol is described to successfully collect and maintain healthy Atta (Hymenoptera: Formicidae) ant colonies in laboratory conditions. Additionally, different nest types and configurations are detailed together with possible experimental procedures.

Collection and Long-Term Maintenance of Leaf-Cutting Ants (Atta) in Laboratory Conditions

Ants are one of the most biodiverse groups of animals on the planet and inhabit different environments. The maintenance of ant colonies in controlled ...

collection-long-term-maintenance-leaf-cutting-ants-atta-laboratory

Research

JoVE Journal

Biology

2.5K Views.  São Paulo State University. Het succesvol verzamelen en onderhouden van kolonies in verschillende soorten nesten onder laboratoriumomstandigheden maakt de ontwikkeling van algemeen onderzoek mogelijk en kan het bereik van de wetenschap bevorderen. Omdat bestand-geteste kolonies moeilijk te controleren zijn, staat dit protocol voorlopige experimentomstandigheden toe in een gecontroleerde omgeving, waarbij hele kolonies als operationele eenheden worden gebruikt. Dit protocol kan worden aangepast aan andere groepen die van...

Video: Collection and Long-Term Maintenance of Leaf-Cutting Ants Atta in Laboratory Conditions

Gibberella zeae Ascospore Production and Collection for Microarray Experiments.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain quality. F. graminearum also causes stalk and ear rots of maize and is a producer of mycotoxins such as the trichothecenes that contaminate grain and are harmful to humans and livestock (Goswami and Kistler, 2004).

The fungus produces two types of spores. Ascospores, the propagules resulting from sexual reproduction, are the main source of primary infection.  These spores are forcibly discharged from mature perithecia and dispersed by wind (Francl et al 1999). Secondary infections are mainly caused by macroconidia which are produced by asexual means on the plant surface. To study the developmental processes of ascospores in this fungus, a procedure for their collection in large quantity under sterile conditions was required. Our protocol was filmed in order to generate the highest level of information for understanding and reproducibility; crucial aspects when full genome gene expression profiles are generated and interpreted. In particular, the variability of ascospore germination and biological activity are dependent on the prior manipulation of the material. The use of video for documenting every step in ascospore production is proposed in order to increase standardization, complying with the increasingly stringent requirements for microarray analysis. The procedure requires only standard laboratory equipment. Steps are shown to prevent contamination and favor time synchronization of ascospores.

To study the developmental processes of ascospores in Gibberella zeae, a procedure for collection under sterile conditions is filmed in order to generate the highest level of information for protocol description. This should facilitate the reproducibility of the experiment, a crucial aspect when full genome expression profile tests are implemented.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain ...

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

Aphids are excellent experimental models for a variety of biological questions ranging from the evolution of symbioses and the development of polyphenisms to questions surrounding insect's interactions with their host plants. Genomic resources are available for several aphid species, and with advances in the next-generation sequencing, transcriptomic studies are being extended to non-model organisms that lack genomes. Furthermore, aphid cultures can be collected from the field and reared in the laboratory for the use in organismal and molecular experiments to bridge the gap between ecological and genetic studies. Last, many aphids can be maintained in the laboratory on their preferred host plants in perpetual, parthenogenic life cycles allowing for comparisons of asexually reproducing genotypes. Aphis nerii, the milkweed-oleander aphid, provides one such model to study insect interactions with toxic plants using both organismal and molecular experiments. Methods for the generation and maintenance of the plant and aphid cultures in the greenhouse and laboratory, DNA and RNA extractions, microsatellite analysis, de novo transcriptome assembly and annotation, transcriptome differential expression analysis, and qPCR verification of differentially expressed genes are outlined and discussed here.

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

The aphid Aphis nerii colonizes on highly-defended plants in the dogbane family (Apocyanaceae) and provides numerous opportunities to study plant-insect interactions. Here, we present a series of protocols for the maintenance of plant and aphid cultures, and the generation and analysis of molecular and -omic data for A. nerii.

Aphids are excellent experimental models for a variety of biological questions ranging from the evolution of symbioses and the development of polyphenisms ...

Genetics

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.

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.

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

Visual Classical Conditioning in Wood Ants

Several species of insects have become model systems for studying learning and memory formation. Although many studies focus on freely moving animals, studies implementing classical conditioning paradigms with harnessed insects have been important for investigating the exact cues that individuals learn and the neural mechanisms underlying learning and memory formation. Here we present a protocol for evoking visual associative learning in wood ants through classical conditioning. In this paradigm, ants are harnessed and presented with a visual cue (a blue cardboard), the conditional stimulus (CS), paired with an appetitive sugar reward, the unconditional stimulus (US). Ants perform a Maxilla-Labium Extension Reflex (MaLER), the unconditional response (UR), which can be used as a readout for learning. Training consists of 10 trials, separated by a 5-minute intertrial interval (ITI). Ants are also tested for memory retention 10 minutes or 1 hour after training. This protocol has the potential to allow researchers to analyze, in a precise and controlled manner, the details of visual memory formation and the neural basis of learning and memory formation in wood ants.

We present a protocol for the classical conditioning of harnessed ants that permits researchers to study visual learning and memory formation at a level of analysis not possible with freely moving individuals.

Several species of insects have become model systems for studying learning and memory formation. Although many studies focus on freely moving animals, ...

Behavior

A Visual Guide for Studying Behavioral Defenses to Pathogen Attacks in Leaf-Cutting Ants

The complex lifestyle, evolutionary history of advanced cooperation, and disease defenses of leaf-cutting ants are well studied. Although numerous studies have described the behaviors connected with disease defense, and the associated use of chemicals and antimicrobials, no common visual reference has been made. The main aim of this study was to record short clips of behaviors involved in disease defense, both prophylactically and directly targeted towards an antagonist of the colony following infection. To do so we used an infection experiment, with sub-colonies of the leaf-cutting ant species Acromyrmex echinatior, and the most significant known pathogenic threat to the ants&#39; fungal crop (Leucoagaricus gongylophorus), a specialized pathogenic fungus in the genus Escovopsis. We filmed and compared infected and uninfected colonies, at both early and more advanced stages of infection. We quantified key defensive behaviors across treatments and show that the behavioral response to pathogen attack likely varies between different castes of worker ants, and between early and late detection of a threat. Based on these recordings we have made a library of behavioral clips, accompanied by definitions of the main individual defensive behaviors. We anticipate that such a guide can provide a common frame of reference for other researchers working in this field, to recognize and study these behaviors, and also provide greater scope for comparing different studies to ultimately help better understand the role these behaviors play in disease defense.

We present a visual guide to disease defense behaviors in leaf-cutting ants, with individual clips and accompanying definitions, illustrated in an experimental infection scenario. Our main aim is to help other researchers recognize key defensive behaviors and to provide a common understanding for future research in this field.

The complex lifestyle, evolutionary history of advanced cooperation, and disease defenses of leaf-cutting ants are well studied. Although numerous studies ...

Preparing and Rearing Axenic Insects with Tissue Cultured Seedlings for Host-Gut Microbiota Interaction Studies of the Leaf Beetle

Insect guts are colonized by diverse bacteria that can profoundly impact the host&#39;s physiological traits. Introducing a particular bacterial strain into an axenic insect is a powerful method to verify gut microbial function and elucidate the mechanisms underlying gut microbe-host interactions. Administering antibiotics or sterilizing egg surfaces are two commonly used methods to remove gut bacteria from insects. However, in addition to the potential adverse effects of antibiotics on insects, previous studies indicated that feeding antibiotics could not eliminate gut bacteria. Thus, germ-free artificial diets are generally employed to maintain axenic insects, which is a tedious and labor-intensive process that cannot fully resemble nutritional components in natural food. Described here is an efficient and simple protocol to prepare and maintain axenic larvae of a leaf beetle&#160;(Plagiodera versicolora). Specifically, surfaces of the beetle eggs were sterilized, following which germ-free poplar leaves were used to rear axenic larvae. The axenic status of the insects was further confirmed via culture-dependent and culture-independent assays. Collectively, by combining egg disinfection and germ-free cultivation, an efficient and convenient method was developed to obtain axenic P. versicolora, providing a readily transferable tool for other leaf-eating insects.

To obtain an axenic insect, its egg surface is sterilized, and the hatched larva is subsequently reared using axenic leaves. This method provides an efficient way for axenic insect preparation without administering antibiotics or developing an artificial diet, which can also be applied to other leaf-eating insects.

Insect guts are colonized by diverse bacteria that can profoundly impact the host&#39;s physiological traits. Introducing a particular bacterial strain ...

Maintaining Laboratory Cultures of Gryllus bimaculatus, a Versatile Orthopteran Model for Insect Agriculture and Invertebrate Physiology

Gryllus bimaculatus (De Geer)&#160;is a large-bodied cricket distributed throughout Africa and Southern Eurasia where it is often wild-harvested as human food. Outside its native range, culturing G. bimaculatus is feasible due to its dietary plasticity, rapid reproductive cycle, lack of diapause requirement, tolerance for high-density rearing, and robustness against pathogens. Thus, G. bimaculatus can be&#160;a versatile model for studies of insect physiology, behavior, embryology, or genetics.
Cultural parameters, such as stocking density, within-cage refugia, photoperiod, temperature, relative humidity, and diet, all impact cricket growth, behavior, and gene expression and should be standardized. In the burgeoning literature on farming insects for human consumption, these crickets are frequently employed to evaluate candidate feed admixtures derived from crop residues, food-processing byproducts, and other low-cost waste streams.
To support ongoing experiments evaluating G. bimaculatus growth performance and nutritional quality in response to variable feed substrates, a comprehensive set of standard protocols for breeding, upkeep, handling, measurement, and euthanasia in the laboratory was developed and is presented here. An industry-standard cricket feed has proven nutritionally adequate and functionally appropriate for the long-term maintenance of cricket breeding stocks, as well as for use as an experimental control feed. Rearing these crickets at a density of 0.005 crickets/cm3 in screen-topped 29.3 L polyethylene cages at an average temperature of 27 &#176;C on a 12 light (L)/12 dark (D) photoperiod, with moistened coconut coir serving both as hydration source and oviposition medium has successfully sustained healthy crickets over a 2-year span. Following these methods, crickets in a controlled experiment yielded an average mass of 0.724 g 0.190 g at harvest, with 89% survivorship and 68.2% sexual maturation between stocking (22 days) and harvest (65 days).

Maintaining Laboratory Cultures of Gryllus bimaculatus, a Versatile Orthopteran Model for Insect Agriculture and Invertebrate Physiology

This paper outlines basic methods to standardize important factors such as density, feed availability, hydration source, and environmental controls for the long-term rearing of laboratory cultures of the edible cricket, Gryllus bimaculatus.

Gryllus bimaculatus (De Geer)&#160;is a large-bodied cricket distributed throughout Africa and Southern Eurasia where it is often wild-harvested as human ...

Bradysia coprophila Lab Protocols and Chromosomal Research

Laboratory Maintenance of the Lower Dipteran Fly Bradysia (Sciara) coprophila: A New/Old Emerging Model Organism

Laboratory stocks of the lower dipteran fly, Bradysia (Sciara) coprophila, have been maintained for over a century. Protocols for laboratory upkeep of B. coprophila are presented here. These protocols will be useful for the rapidly increasing number of laboratories studying B. coprophila to take advantage of its unique biological features, which include (1) a monopolar spindle in male meiosis I; (2) non-disjunction of the X dyad in male meiosis II; (3) chromosome imprinting to distinguish maternal from paternal homologs; (4) germ line-limited (L) chromosomes; (5) chromosome elimination (paternal chromosomes in male meiosis I; one to two X chromosomes in early embryos; L chromosomes from the soma in early embryos); (6) sex determination by the mother (there is no Y chromosome); and (7) developmentally regulated DNA amplification at the DNA puff loci in larval salivary gland polytene chromosomes. 
It is now possible to explore these many unique features of chromosome mechanics by using the recent advances in sequencing and assembly of the B. coprophila genome and the development of transformation methodology for genomic engineering. The growing scientific community that uses B. coprophila for research will benefit from the protocols described here for mating the flies (phenotypic markers for mothers that will have only sons or only daughters; details of mass mating for biochemical experiments), checking embryo hatch, feeding larvae, and other comments on its rearing.

Author Spotlight: Exploring Bradysia coprophila's Unique Biology &#8211; A Guide to Laboratory Maintenance

This paper outlines the laboratory maintenance (including mating and feeding) of the lower dipteran fly Bradysia (Sciara) coprophila.

Laboratory stocks of the lower dipteran fly, Bradysia (Sciara) coprophila, have been maintained for over a century. Protocols for laboratory upkeep of B. ...

 Microscale SEM Analysis of Microbial Communities in Fungus-Growing Ant Cultures

Microbiota of Attine Ants' Gardens: Visualizing a Microbial Landscape by Scanning Electron Microscopy

In macroscale ecosystems, such as rainforests or coral reefs, the spatial localization of organisms is the basis of our understanding of community ecology. In the microbial world, likewise, microscale ecosystems are far from a random and homogeneous mixture of organisms and habitats. Accessing the spatial distribution of microbes is fundamental for understanding the functioning and ecology of the microbiota, as cohabiting species are more likely to interact and influence each other&#39;s physiology.
An interkingdom microbial ecosystem is at the core of fungus-growing ant colonies, which cultivate basidiomycete fungi as a nutritional resource. Attine ants forage for diverse substrates (mostly plant-based), metabolized by the cultivated fungus while forming a spongy structure, a &#34;microbial garden&#34; that acts as an external gut. The garden is an intertwined mesh of fungal hyphae growing by metabolizing the substrate, opening niches for a characteristic and adapted microbiota to establish. The microbiota is thought to be a contributor to substrate degradation and fungal growth, though its spatial organization is yet to be determined.
Here, we describe how we employ Scanning Electron Microscopy (SEM) to investigate, with unprecedented detail, the microbiota and biofilm spatial organization across different fungiculture systems of fungus-growing ants. SEM imaging has provided a description of the microbiota spatial structure and organization. SEM revealed that microbiota commonly assemble in biofilms, a widespread structure of the microbial landscapes in fungiculture. We present the protocols employed to fix, dehydrate, dry, sputter coating, and image such a complex community. These protocols were optimized to deal with delicate and heterogeneous samples, comprising plant and fungal biomass, as well as the microbiota and the biofilm.

We propose an optimized Scanning Electron Microscopy protocol for visualizing highly heterogeneous and delicate samples containing plant and fungal biomass, together with microbiota and biofilm. This protocol allows describing the spatial dimensions of the microbiota organization.

Collection and Long-Term Maintenance of Leaf-Cutting Ants (Atta) in Laboratory Conditions

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