nematode worm culture in dog food agar plates for behavioral analysis

חקירת התנהגויות קולקטיביות בנמטודות באמצעות מדיום אגר מזון לכלבים

Both nonscientists and scientists are fascinated with animals&#39; collective behaviors, as in flocks of birds and schools of fish. Collective behaviors have been analyzed in a broad range of fields, including physics, biology, mathematics, and robotics. In particular, active matter physics is a growing research field that focuses on systems composed of self-propelled elements, that is, dissipative systems, such as flocks of birds, schools of fish, biofilms of motile bacteria, cytoskeletons composed of active molecules, and groups of self-propelled colloids. The theory of active matter physics maintains that however complex the behaviors of individuals are, the collective motions of enormous numbers of living things are governed by a small number of simple rules. For example, the Vicsek model, a candidate for a unified description of the collective motion of self-propelled particles, predicts that short-range alignment interaction of moving objects is required to form a long-range ordered phase with eccentric fluctuation in 2D, as in herds of animals1. Top-down experimental approaches pertaining to the physics of active matter are developing rapidly. Previous experiments confirmed the formation of a long-range ordered phase in Escherichia coli2. Other recent works&#160;employed cells3,4, bacteria5, motile colloids6, or moving proteins7,8. Simple minimal models such as the Vicsek model successfully described these real phenomena. In contrast with these unicellular experimental systems, collective behaviors by animals are usually observed in the wild, as no one could hope to perform controlled experiments with 10,000 real birds or fish.
Biologists share the same interest as physicists: how individuals interact with each other and functionally behave as a group. One of the traditional research fields for analyzing individual behavior is neuroscience, in which the mechanisms underlying behavior have been examined at the neuronal and molecular levels. Many neuroscientific bottom-up approaches have been developed thus far. Top-down approaches in physics and bottom-up approaches in biology can be facilitated using model animals such as the fruit fly, the worm Caenorhabditis elegans, and the mouse9. However, there have been few findings on the large-scale collective behavior of these model animals in the laboratory10; it is still difficult to prepare genetically tractable model animals on a large scale in the laboratory. Therefore, in current research on collective behaviors in biology and physics, it has been difficult for scientists who usually do research in the laboratory to study animals&#39; collective behaviors.
In this study, we established a method for the large-scale cultivation of nematodes to study their collective behaviors. This system allows us to change environmental conditions and examine the effect of individual-level locomotion on collective behaviors using mutants10. In active matter physics, the parameters of the mathematical model can be controlled in both experiments and simulations, which enables verification of that model for identifying unified descriptions. Genetics is used to understand the neural circuit mechanism underlying collective behavior11.

מחבר זרקור: ניתוח התנהגותי קולקטיבי של הנמטודה, Caenorhabditis elegans

טיפוח בקנה מידה גדול של נמטודות כדי ללמוד את ההתנהגויות הקולקטיביות שלהם

We are studying the mechanism underlying collective behavior from the perspective of active metaphysics and neuroscience. In the research field of collective behavior, experimental system has been established using non-living things and at the cellular level. However, it remains still challenging to design the animal-level experimental system to study the collective behavior in the laboratory.We successfully reconstructed the collective behavior with traditionally used model animal, nematode C.elegans. This system make it possible to control the parameter with mathematical model proposed for animals'collective behavior, and studies the neuromechanism underlying collective behavior using molecular genetics.

תרבית תולעי נמטודה במזון כלבים צלחות אגר לניתוח התנהגותי

התחילו בהנחת ארבע תולעים בוגרות על מצע גידול נמטודות או צלחת אגר NGM עם זרעי E.coli OP50. לגדל את הדור הראשון של התולעים לרעב על צלחת NGM בטמפרטורה של 23 מעלות צלזיוס במשך שבעה ימים. לאחר מכן, העבירו כמויות קטנות של אגר מזון כלבים או מדיום DFA למרכז צלחת NGM חדשה עם E.coli OP50.לניסויים אופטוגנטיים, יש לשפוך 40 מיקרוליטר של כל-טרנס-רטינל על DFA לפני חיסון התולעים. במקום הבא, 0.5 מיליגרם של מזון כלבים על מדיום DFA, במרחק של כשני מילימטרים ממכסה הצלחת. הימנע מזיהום על ידי הקרנת צלחת NGM באור אולטרה סגול למשך 15 דקות.אספו את התולעים המורעבות מצלחות ה-NGM באמצעות מים מעוקרים. לאחר מכן חסנו את התולעים שנאספו על תווך DFA הממוקם על הגז הטבעי הנוזלי. להפיץ את התולעים ב 23 מעלות צלזיוס, המאפשר להם לטפס לכיוון מכסה הצלחת במשך 10 עד 14 ימים.לחות מוגברת הציגה גדלי תאים גדולים יותר של תבניות רשת התולעים לפני שבסופו של דבר קרסה, והותירה אשכולות תולעים רדומים על פני השטח הפנימיים של המכסה.

nematode-worm-culture-in-dog-food-agar-plates-for-behavioral-analysis

Research

JoVE Journal

Biology

Nematode Worm Culture in Dog Food Agar Plates for Behavioral Analysis

52 Views.  Hiroshima University. התחילו בהנחת ארבע תולעים בוגרות על מצע גידול נמטודות או צלחת אגר NGM עם זרעי E.coli OP50. לגדל את הדור הראשון של התולעים לרעב על צלחת NGM בטמפרטורה של 23 מעלות צלזיוס במשך שבעה ימים. לאחר מכן, העבירו כמויות קטנות של אגר מזון כלבים או מדיום DFA למרכז צלחת NGM חדשה עם E.coli OP50.לניסויים אופ...

Video: תרבית תולעי נמטודה במזון כלבים צלחות אגר לניתוח התנהגותי

The Large-Scale Cultivation of Nematodes to Study Their Collective Behaviors

Animals exhibit dynamic collective behaviors, as observed in flocks of birds, schools of fish, and crowds of humans. The collective behaviors of animals have been investigated in the fields of both biology and physics. In the laboratory, researchers have used various model animals such as the fruit fly and zebrafish for approximately a century, but it has remained a major challenge to study large-scale complex collective behavior orchestrated by these genetically tractable model animals. This paper presents a protocol to create an experimental system of collective behaviors in Caenorhabditis elegans. The propagated worms climb on the lid of the Petri plate and show collective swarming behavior. The system also controls worm interactions and behaviors by changing the humidity and light stimulation. This system allows us to examine the mechanisms underlying collective behaviors by changing environmental conditions and examining the effects of individual-level locomotion on collective behaviors using mutants. Thus, the system is useful for future research in both physics and biology.

Here, a system is reported for studying the collective behaviors of nematodes by culturing them in bulk using dog food agar medium. This system allows researchers to propagate large numbers of dauer worms and can be applied to Caenorhabditis elegans and other related species.

Animals exhibit dynamic collective behaviors, as observed in flocks of birds, schools of fish, and crowds of humans. The collective behaviors of animals ...

Begin by placing four adult worms on an nematode growth medium or NGM agar plate seeded with E.coli OP50. Grow the first generation worms to starvation on the NGM plate at a temperature of 23 degrees Celsius for seven days. Next, transfer small amounts of dog food agar or DFA medium to the center of a new NGM plate seeded with E.coli OP50.For optogenetic experiments, pour 40 microliters of all-trans-retinal onto the DFA prior to worm inoculation. Next place, 0.5 milligrams of dog food on the DFA medium, about two millimeters away from the plate lid. Avoid contamination by irradiating the NGM plate with ultraviolet light for 15 minutes.Gather the starved worms from the NGM plates using sterilized water. Then inoculate the gathered worms onto the DFA medium located on the NGM. Propagate the worms at 23 degrees Celsius, enabling them to climb towards the plate lid over 10 to 14 days.Increased humidity exhibited larger compartment sizes of the worm network patterns before ultimately collapsing, leaving dormant worm clusters on the inner surface of the lid.

Observation of the Collective Behavior and Optogenetic Experimentation of Nematode Dauer Worms

Begin by placing a new NGM plate devoid of E.coli and DFA onto an aluminum plate On the stage of a macro zoom microscope. Use a Peltier temperature controller unit to maintain the plate bottom at 23 degrees Celsius temperature for at least five minutes. Next, replace the plate with the lid of a plate that the nematode worms have climbed up onto.Increase the temperature of the plate bottom to 26 degrees Celsius to change the interior humidity. Now, capture images of the inner surface of the plate lid at 20 frames per second using the microscope camera, keep the ZX899 worms on DFA, maintaining this condition for five minutes before illumination. Now illuminate the ZX899 worms attached to a Petri plate lid, maintained at 23 degrees Celsius.Capture the images of the inner plate surface at 20 frames per second. Increased humidity exhibited larger compartment sizes of the worm network patterns before ultimately collapsing, leaving dormant worm clusters on the inner surface of the lid.