The authors thank Drs. Jeffrey Bloomquist and Daniel Swale for their technical comments and suggestions. This project was partially funded by the Department of Entomology and the College of Agriculture and Life Sciences at Virginia Tech.

1. Recogida y preparación de los sujetos de prueba <ol><li> Recoger el número apropiado de las abejas de la colonia. NOTA: El número necesario depende no sólo del tamaño y alcance del experimento, sino también la habilidad del investigador. Por ejemplo, si hay 2 grupos de tratamiento con un tamaño deseado de la muestra de 10 abejas por grupo, un investigador experto razonablemente podría recoger un mínimo de 30 abejas para dar cuenta de las disecciones sin éxito y terminar con 20 videos útiles para marcar. </li><li> Minimizar la cantidad de tiempo que transcurre entre la recolección y la disección. NOTA: A pesar de abejas pueden ser alojados en el laboratorio para día antes de la disección, la tasa de éxito de las disecciones (es decir, la probabilidad de que el mantenimiento de un ritmo cardíaco estable en un vaso dorsal diseccionado) se ha observado para disminuir con relación a la cantidad de tiempo que las abejas se encuentran fuera de la colonia. <ol><li> Proporcionar abejas con una fuente de agua y alimentos, mientras alojados en el laboratorio. para example, como mínimo, proporcionar acceso a un 50% de solución de sacarosa en agua (w / v) (esto es suficiente para duraciones de menos de 6 h). Para períodos más largos, proporcionan acceso a las abejas de miel. </li><li> abejas de la casa de la noche a la mañana de laboratorio a una temperatura de aproximadamente 32 ° C y 60-80% de humedad relativa para reducir el estrés y evitar la deshidratación. </li></ol></li><li> Antes de la disección, anestesiar abejas brevemente para ayudar en la manipulación. NOTA: Esto puede disminuir la tasa de éxito de las disecciones y reducir el rendimiento. <ol><li> Enfriar las abejas ya sea colocándolos en hielo o en un refrigerador durante el tiempo suficiente para reducir el movimiento con el fin de ayudar en el manejo. </li><li> Alternativamente, exponer brevemente las abejas a CO 2 con el fin de ayudar en el manejo. NOTA: La exposición prolongada al frío puede reducir la tasa de éxito de las disecciones. Extendido o repetida exposición al CO 2 también puede reducir la tasa de éxito de las disecciones. </li></ol></li></ol> 2. DisecciónDorsal de la pared abdominal NOTA: Las abejas deben estar vivos en el momento de la disección. <ol><li> El uso de pinzas y / o tijeras de microdisección, eliminar patas y las alas para facilitar la disección del abdomen. Mantenga un pequeño vaso de precipitados o recipiente similar lleno de agua destilada cercano con el fin de enjuagar los instrumentos entre disecciones. </li><li> Mientras que restringir la abeja con fórceps, utilizar las tijeras de microdisección para cortar lateralmente a lo largo de la pared abdominal dorsal entre la primera y segunda tergites (véase la Figura 1). </li></ol><img alt="Figura 1" src="/files/ftp_upload/55029/55029fig1.jpg" /> Figura 1: Vista dorsal del abdomen de la abeja. La incisión inicial debe hacerse entre la primera y segunda tergites, como se indica mediante la línea roja. Barra de escala = 1 mm. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55029/55029fig1large.jpg" target="_blank">Por favor, haga clic en ella</a>e para ver una versión más grande de esta figura. <ol start="3"><li> Aunque ligeramente agarrando el borde posterior de la segunda Tergito con las pinzas, corta longitudinalmente a lo largo de cada lado de la abeja de la incisión inicial a la aguijón (ver Figura 2). Tenga cuidado al cortar para evitar perforar el tubo digestivo. </li></ol><img alt="Figura 2" src="/files/ftp_upload/55029/55029fig2.jpg" /> Figura 2: Vista lateral del abdomen de la abeja. El segundo y tercer incisiones deben hacerse a lo largo de cada lado del abdomen, como se indica mediante la línea roja. Barra de escala = 1 mm. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55029/55029fig2large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> <ol start="4"><li> Intercambiar las tijeras por un segundo juego de pinzas finas y utilizarlos para separar cuidadosamente la pared abdominal dorsal del resto of abdomen. Retire con cuidado el aguijón y cualquier parte del tracto gastrointestinal que permanece unido a la pared abdominal dorsal. Evitar la ruptura de la tripa, ya que los contenidos pueden recubrir la pared abdominal y dificultan visualización del vaso dorsal. </li></ol><img alt="figura 3" src="/files/ftp_upload/55029/55029fig3.jpg" /> Figura 3: Vista del vaso dorsal. Una vez que el intestino y aguijón se han eliminado, el vaso dorsal es visible a lo largo de la línea media de la pared abdominal dorsal diseccionado. Barra de escala = 1 mm. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55029/55029fig3large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> <ol start="5"><li> Organizar la pared abdominal dorsal en la orientación deseada por debajo de la cámara para que el vaso dorsal es visible (véase la Figura 3). Utilizar las tijeras de microdisección para recortar cualquier exceso de abdominal pared que impide la visualización del vaso dorsal. La forma de la pared abdominal dorsal debe ser similar a una taza o recipiente poco profundo cuando situada correctamente. <ol><li> Desde el vaso dorsal no se extiende en el segmento abdominal retaguardia de la abeja, quitar el Tergito final con el fin de mejorar la visualización del vaso dorsal. </li></ol></li><li> Utilizando una micropipeta de volumen ajustable, cubrir el vaso dorsal con 10 l de una solución isotónica para mantener las condiciones fisiológicas y facilitar un constante latido del corazón. NOTA: La solución recomendada es la fuerza trimestre solución de Ringer (0,120 g / l de cloruro de calcio, 0.105 g / L de cloruro de potasio, 0,050 g / L de bicarbonato de sodio, y 2,250 g / L de cloruro de sodio), que se ha encontrado para facilitar un establo, latido continuo. </li></ol> 3. La observación y la modulación del ritmo cardíaco <ol><li> Permitir que el vaso dorsal permanezca inmóvil hasta que se logre un pulso continuo estable(Normalmente dentro de 300 segundos). NOTA: El latido del corazón es visualizado como contracciones rítmicas del vaso dorsal. Inicialmente, puede parecer que no hay latido del corazón, especialmente si la abeja fue anestesiado, pero el corazón latiendo generalmente reanudar después de descansar en solución isotónica durante varios minutos y puede seguir batiendo durante horas, siempre que sigan siendo bañado en solución. </li><li> Medir la frecuencia cardíaca en términos del número de latidos por minuto (BPM). <ol><li> Registrar el número de contracciones observadas durante un periodo de 60 seg. Utilizar un contador de cuenta de la mano y un temporizador para facilitar este proceso. </li></ol></li><li> Medir el cambio en la frecuencia cardíaca mediante el registro de los BPM observado antes y después del tratamiento con un compuesto cardiomodulatory. NOTA: Aunque el tiempo necesario para observar un efecto sobre la frecuencia cardíaca puede variar dependiendo del compuesto que se está probando, los cambios en la frecuencia cardíaca normalmente se pueden observar dentro de minutos. <ol><li> Determinar la frecuencia cardíaca basal inmediatamente antes dela adición de cualquier compuesto de prueba. NOTA: la frecuencia cardiaca post-tratamiento por lo general se puede determinar después de 90 a 120 seg. </li><li> Preparar potenciales cardiomodulators (por ejemplo, octopamina) disolviendo el compuesto en la misma solución isotónica usada para bañar al vaso dorsal. </li><li> Añadir los compuestos de ensayo a la solución que rodea el vaso dorsal mediante la utilización de una micropipeta. </li></ol></li><li> Para una mayor precisión y un mayor rendimiento, una grabación de vídeo de cada sujeto de prueba y luego usar los videos para marcar el ritmo cardíaco en un momento posterior. NOTA: Esto permite que un solo investigador escalonar disecciones con el fin de facilitar la producción casi continua de videos. Cuando la grabación de vídeos, la longitud mínima recomendada es de aproximadamente 240 segundos con cualquier compuesto de ensayo que se añade en la marca de 60 seg. Esto asegura que el investigador tiene una ventana de 60 seg para asegurar la frecuencia cardíaca basal y luego otra ventana 60 seg para asegurar la frecuencia cardíaca post-tratamiento 120 secdespués del tratamiento. </li></ol>

<ol>
	<li>Klowden, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulatory+Systems.">Circulatory Systems.</a> Physiological Systems in Insects, 3rd Edition. , 365-413 (2013).</li><li>League, G. P., Onuh, O. C., Hillyer, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+structural+and+functional+analysis+of+the+larval+and+adult+dorsal+vessel+and+its+role+in+hemolymph+circulation+in+the+mosquito+Anopheles+gambiae.">Comparative structural and functional analysis of the larval and adult dorsal vessel and its role in hemolymph circulation in the mosquito Anopheles gambiae.</a> J Exp Biol. 218 (Pt 3), 370-380 (2015).</li><li>Wasserthal, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillating+Hemolymph+Circulation+in+the+Butterfly+Papilio-Machaon+L+Revealed+by+Contact+Thermography+and+Photocell+Measurements.">Oscillating Hemolymph Circulation in the Butterfly Papilio-Machaon L Revealed by Contact Thermography and Photocell Measurements.</a> J Comp Physiol. 139 (2), 145-163 (1980).</li><li>Wasserthal, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+circulation+and+tracheal+ventilation+in+holometabolous+insects.">Interaction of circulation and tracheal ventilation in holometabolous insects.</a> Adv Insect Physiol. 26, 297-351 (1996).</li><li>Schwab, E. R., Chilson, R. A., Eddleman, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heartbeat+Rate+Modulation+Mediated+by+the+Ventral+Nerve+Cord+in+the+Honey-Bee,+Apis-Mellifera.">Heartbeat Rate Modulation Mediated by the Ventral Nerve Cord in the Honey-Bee, Apis-Mellifera.</a> J Comp Physiol B-Biochem Syst Environ Physiol. 161 (6), 602-610 (1991).</li><li>Kaiser, W., Weber, T., Otto, D., Miroschnikow, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+supply+of+the+heart+and+electrocardiogram+potentials+with+reversed+polarity+in+sleeping+and+resting+honey+bees.">Oxygen supply of the heart and electrocardiogram potentials with reversed polarity in sleeping and resting honey bees.</a> Apidologie. 45 (1), 73-87 (2014).</li><li>Papaefthimiou, C., Theophilidis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Octopamine--a+single+modulator+with+double+action+on+the+heart+of+two+insect+species+(Apis+mellifera+macedonica+and+Bactrocera+oleae):+Acceleration+vs.+inhibition.">Octopamine--a single modulator with double action on the heart of two insect species (Apis mellifera macedonica and Bactrocera oleae): Acceleration vs. inhibition.</a> J Insect Physiol. 57 (2), 316-325 (2011).</li><li>Castro, C. A., Hogan, J. B., Benson, K. A., Shehata, C. W., Landauer, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral-Effects+of+Vehicles+-+Dmso,+Ethanol,+Tween-20,+Tween-80,+and+Emulphor-620.">Behavioral-Effects of Vehicles - Dmso, Ethanol, Tween-20, Tween-80, and Emulphor-620.</a> Pharmacol Biochem Behav. 50 (4), 521-526 (1995).</li><li>Papaefthimiou, C., Papachristoforou, A., Theophilidis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biphasic+responses+of+the+honeybee+heart+to+nanomolar+concentrations+of+amitraz.">Biphasic responses of the honeybee heart to nanomolar concentrations of amitraz.</a> Pestic Biochem Phys. 107 (1), 132-137 (2013).</li><li>Roeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Octopamine+in+invertebrates.">Octopamine in invertebrates.</a> Prog Neurobiol. 59 (5), 533-561 (1999).</li><li>Johnson, E., Ringo, J., Dowse, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+Drosophila+heartbeat+by+neurotransmitters.">Modulation of Drosophila heartbeat by neurotransmitters.</a> J Comp Physiol B. 167 (2), 89-97 (1997).</li></ol>

The authors have nothing to disclose.

The protocol presented here provides a simple and effective approach to testing pharmacological compounds for their effects on honey bee heart rate. As observed in prior experiments that either transect the ventral nerve cord of a living insect5 or dissect out the ventral nerve cord when exposing the dorsal vessel7, the loss of central nervous system regulation results in a stable, low frequency heartbeat. The low frequency of beats allows the investigator to visually assess heart rate without having to rely on delicate or expensive instrumentation. As has been demonstrated, the solvent DMSO can be used in low concentrations to dissolve pharmacological agents for delivery via the isotonic solution used to bath the dorsal vessel. Furthermore, heart rate can be accelerated in this method using the invertebrate neuromodulator octopamine, which has been previously shown to act as a cardioaccelerant in bees7. Although this protocol was designed in order to measure changes in heart rate, it is possible to use it to observe other, more qualitative characteristics of heartbeat as well, such as changes in the pattern of contractions. Regardless of the end point in question, the protocol relies primarily upon the ability of the investigator to perform a clean, rapid dissection. The most common challenge that that an investigator will face when using this protocol is the failed dissection, which results in a dissected abdominal wall that is not useful either because there is no heartbeat, or because the dorsal vessel is not visible.
The dorsal vessel can be easily obscured as the result of a poor dissection. Puncturing or tearing the gut can result in gut contents spilling out onto the abdominal wall, typically in the form of yellow/brown excrement that mixes with the vehicle and obscures the dorsal vessel. Although the abdominal wall can be rinsed with an isotonic solution to remove debris, doing so can increase the chances of damaging the dorsal vessel. With practice, this can be easily avoided; however, if this remains an issue, an alternative is to remove the gut prior to dissection. This can be accomplished by first removing the stinger and then pulling the abdominal gastrointestinal tract out through the same hole. One noticeable difference between this approach and the dissection as described in the protocol is that removing the stinger and gut first will typically leave the dorsal abdominal wall covered by a translucent membrane that can also obscure the dorsal vessel. When dissecting the abdomen as described in the protocol, this membrane will usually be removed along with the gut. If the membrane, or some portion of it, is obstructing view, it can be removed carefully with fine forceps. 
It is not uncommon for the dorsal vessel to display no sign of a heartbeat immediately following dissection. Bathing it in an isotonic solution and allowing it to rest for several minutes will usually result in a steady heartbeat resuming. A continued lack of a heartbeat can be the result of a number of different issues, including damage to the dorsal vessel caused by a clumsy dissection, excessive anesthesia, premature death of the bee, or a slow dissection. The most common cause, however, would seem to be related to the energy reserves of the bee prior to dissection. Bees that have been housed in the laboratory overnight, even when provided access to food and water, are far more likely to result in a failed dissection than bees just collected from the colony. Age may also be a factor, as the success rate appears to be greater with nurse bees than with foragers. This has not been tested in a controlled study, however, and would be an interesting line of inquiry to pursue.
Overall, the goal of this protocol is to provide a simple, inexpensive approach to assessing the effect of potential cardiomodulators on bee heart rate. This protocol could be adapted to observe differences in cardiac function as a result of other types of treatments or factors, or for use with other insects. The greatest limitation of the protocol is that the dorsal vessel is not being observed in a living, intact insect. It is likely that some potentially cardiomodulatory compounds will not show an effect on heart rate if they function at the level of the central nervous system. In this case, an electrophysiological approach is likely necessary. The greatest strength of this protocol is its simplicity, as it costs little, requires little equipment, can be undertaken with little or no specialized training, and can be conducted quickly once mastered. As such, it can be a valuable tool for applications ranging from exploratory pilot work to large scale drug screening studies, and can also serve as a useful teaching tool in high school or university classrooms.

El objetivo general de esta metodología es permitir que el investigador para observar y cuantificar el efecto que tiene un agente farmacológico en el ritmo cardíaco de las abejas melíferas forma rápida y sencilla. Las abejas, como otros insectos, tienen un sistema circulatorio abierto que difunde hemolinfa, el equivalente de insectos de la sangre, a través de la cavidad del cuerpo, conocida como la hemocoel. La circulación de hemolinfa es esencial para el transporte de nutrientes, factores inmunológicos, productos de desecho, así neurohormonas y otras moléculas de señalización 1. Circulation es facilitado por el vaso dorsal, que se extiende a lo largo de la línea media dorsal del insecto, así como los órganos pulsátiles accesorios. El vaso dorsal se divide en dos secciones funcionalmente distintas, designado el corazón en el abdomen y la aorta en el tórax y la cabeza. contracciones propagadas en la hemolinfa corazón bombee hacia el tórax y la cabeza, mientras que los órganos pulsátiles accesorios garantizan el flujo de la hemolinfa de las extremidades. <pclass = &quot;jove_content&quot;&gt; función cardíaca de insectos se puede observar usando una variedad de métodos, dependiendo de la magnitud, fase fisiología, o la vida del insecto. Un enfoque común para la observación de la frecuencia cardíaca en larvas o insectos más pequeños es el uso de imágenes intravital 2. Este método es menos útil en las abejas adultas, sin embargo, ya que puede ser difícil de ver claramente el vaso dorsal a través de la pared abdominal. Un enfoque establecido para el registro de la frecuencia cardíaca en una variedad de insectos, incluyendo abejas, es el uso de la termografía de contacto, que utiliza termistores aplicado en el exterior del insecto para detectar pulsaciones cardiacas 3,4. La frecuencia cardíaca en las abejas adultas también ha sido grabado utilizando una técnica electrofisiológica para medir una señal de impedancia eléctrica de 4,5. Esta técnica requiere la inserción de electrodos en el animal al lado del corazón y el uso de un convertidor de impedancia para registrar los latidos del corazón 4. Del mismo modo, electrocardiogramas se han utilizado para DETECt señales eléctricas producidas por el corazón y combinados con grabación de vídeo de la abeja para observar los cambios en la actividad cardíaca 6. Una clara ventaja de estos enfoques es que la frecuencia cardíaca se evalúa en una, abeja vivas intactas, en lugar de en un espécimen disecado, que ayuda a asegurar la disponibilidad de toda la gama de respuestas fisiológicas en el tema. Los desafíos de estos enfoques incluyen la contabilidad para la inmovilización o la anestesia de la materia, la necesidad de limitar las variables externas y estímulos que podrían alterar la frecuencia cardiaca, así como la determinación de un método de entrega adecuado al probar agentes farmacológicos. Otro enfoque que se ha utilizado para el estudio de la actividad cardiaca de abeja es para diseccionar parcialmente el insecto con el fin de exponer el corazón, a continuación, medir contracciones vaso dorsal utilizando un transductor de desplazamiento de fuerza 7. En este protocolo, el corazón se baña continuamente con solución salina fisiológica en marcha y prueba de compounds se pueden disolver en esta solución para su aplicación al objeto 7. Una diferencia significativa entre este método y las descritas anteriormente es que el cordón nervioso ventral se elimina, eliminando el papel que el sistema nervioso central se ha demostrado que desempeña en la modulación de la frecuencia cardíaca 5. El resultado es que el latido del corazón de referencia, que suele ser bastante irregular, se estabiliza a una frecuencia mucho más baja y amplitud que se observa típicamente en un insecto que viven 5,7. Lo que todos estos métodos tienen en común es que requieren un equipo altamente especializado y, a menudo caro, además de un cierto nivel de experiencia, con el fin de llevar a cabo. Tal vez la mayor desventaja es que ninguno de estos enfoques son particularmente adecuados para experimentos que involucran el examen de un gran número de temas, tales como selección de una biblioteca de compuestos potencialmente cardiomodulatory. La mayor fortaleza del método descrito aquíes su simplicidad. El protocolo es relativamente fácil de dominar, la instalación requiere poco espacio, y sólo una entrada financiero mínimo es necesario. El método requiere poco más que algunas abejas, algunos instrumentos quirúrgicos, una solución isotónica, y, o bien un microscopio digital o un microscopio tradicional con una cámara digital. Las abejas son diseccionados para visualizar el vaso dorsal y vídeos digitales se utilizan para registrar la frecuencia cardíaca antes y después del tratamiento con agentes farmacológicos. Aunque la grabación de vídeo no es realmente necesario para observar los cambios en el ritmo cardíaco, además de aumentar considerablemente el rendimiento (es decir, el número de sujetos que se pueden procesar en un período de tiempo determinado). El investigador puede maximizar la eficiencia mediante el registro de un gran número de vídeos a la vez y, posteriormente, marcando estos videos en un momento más conveniente. Otra ventaja de este enfoque es que los vídeos permiten al investigador a empezar de nuevo, se debe interrumpir el proceso de puntuación, y que sea más fácil para el vdel Visor para cegar al tratamiento con el fin de reducir el sesgo.

<table border="0" cellpadding="0" cellspacing="0" width="834">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:200px;">Dino-Lite Edge digital USB microscope</td>
			<td style="width:111px;">Dino-Lite</td>
			<td style="width:119px;">AM4815ZT</td>
			<td style="width:405px;">Any digital microscope or similar setup will suffice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:200px;">Microscope stand</td>
			<td style="width:111px;">Dino-Lite</td>
			<td style="width:119px;">RK-10</td>
			<td>Any stand appropriate for the digital microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:200px;">Laptop or PC</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td>Necessary for digital microscope</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:200px;">Microdissection scissors (Vannas, 8cm, Straight, 5mm Blades)</td>
			<td style="width:111px;">World Precision Instruments</td>
			<td style="width:119px;">14003</td>
			<td>Any similar scissors suitable for microdissection will suffice</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:200px;">Microdissecting Forceps, 10.2cm, Angled (2 pair)</td>
			<td style="width:111px;">World Precision Instruments</td>
			<td style="width:119px;">504482</td>
			<td>Any similar forceps suitable for microdissection will suffice</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:200px;">Ringers solution 1/4 strength tablets&#160;</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">96724-100TAB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:200px;">Dissecting tray</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td>Any surface suitable for microdissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:200px;">Single channel 10 &#181;l pipette</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td>Any device capable of accurately delivering 10 &#181;l volume</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:200px;">Pipette tips</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:200px;">Small beaker or container of water</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td>Used to rinse instruments between subjects</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:200px;">Hand tally counter</td>
			<td style="width:111px;">Office Depot</td>
			<td style="width:119px;">295033</td>
			<td>Any similar product will suffice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:200px;">Timer</td>
			<td style="width:111px;">Office Depot</td>
			<td style="width:119px;">644219</td>
			<td>Any similar product will suffice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:200px;">Deionized water</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td>Preparation of Ringers solution and rinsing instruments</td>
		</tr>
	</tbody>
</table>

dissection and observation of honey bee dorsal vessel for studies of cardiac function

The European honey bee, Apis mellifera L., is a valuable agricultural and commercial resource noted for producing honey and providing crop pollination services, as well as an important model social insect used to study memory and learning, aging, and more. Here we describe a detailed protocol for the dissection of the dorsal abdominal wall of a bee in order to visualize its dorsal vessel, which serves the role of the heart in the insect. A successful dissection will expose a functional heart that, under the proper conditions, can maintain a steady heartbeat for an extended period of time. This allows the investigator to manipulate heart rate through the application of cardiomodulatory compounds to the dorsal vessel. By using either a digital microscope or a microscope equipped with a digital camera, the investigator can make video recordings of the dorsal vessel before and after treatment with test compounds. The videos can then be scored at a time convenient to the user in order to determine changes in heart rate, as well as changes in the pattern of heartbeats, following treatment. The advantages of this protocol are that it is relatively inexpensive to set up, easy to learn, requires little space or equipment, and takes very little time to conduct.

Dado que muchos de los compuestos farmacológicamente activos que pueden ser probados usando este protocolo no son solubles en agua, es necesario tener un disolvente fiable que permitirá a los compuestos de ensayo para ser entregados a través de la solución isotónica usada para bañar al vaso dorsal. Dimetil sulfóxido (DMSO) es un disolvente que se utiliza comúnmente como un vehículo para la administración de fármacos experimentales y otros compuestos en los animales 8, y se ha utilizado con éxito para este propósito en los estudios que examinan el efecto de los pesticidas en la actividad cardíaca abeja de la miel 9. En consecuencia, DMSO fue seleccionado como un vehículo potencial para la entrega de los compuestos de ensayo en el recipiente de dorsal, que requiere que primero se ensayó la actividad cardiomodulatory. En este protocolo, el vaso dorsal diseccionado es bañado inicialmente en 10 l de solución al cuarto de Ringer y la frecuencia cardíaca se determina antes de añadir los compuestos de ensayo. Los compuestos de ensayo are entregado a una concentración conocida en 10 l de DMSO disueltos en solución al cuarto de Ringer y luego se observa el vaso dorsal para los cambios posteriores en la frecuencia cardíaca. Antes de probar cualquier compuesto, un intervalo de concentración de DMSO fue probado para cualquier efecto sobre la frecuencia cardíaca, con relación a la solución de fuerza trimestre de Ringer con 0% de DMSO. DMSO se disolvió a 0,2%, 2%, 10% y 20% v / v en solución al cuarto de Ringer con el fin de probar una concentración final de 0,1%, 1%, 5% y 10% cuando se añade en un volumen igual a la solución ya bañando el vaso dorsal. Este enfoque se utilizó, en lugar de la adición de DMSO a la concentración deseada en los 10 l iniciales utilizados para bañar al vaso dorsal, porque no todos los disección produce un latido del corazón estable. Si DMSO tiene un efecto adverso sobre la frecuencia cardiaca, este efecto podría no ser distinguible de una disección fallado. La frecuencia cardíaca se evaluó inmediatamente antes dela adición de DMSO, y luego de nuevo 120 seg después. La Figura 4 muestra los resultados de este experimento, lo que demuestra que 0,1% y 1% de DMSO en la solución no tuvo ningún efecto significativo sobre la frecuencia cardiaca, con relación a la fuerza trimestre solución de Ringer sin DMSO, mientras que 5% y 10% de DMSO se redujo significativamente la frecuencia cardiaca. <img alt="Figura 4" src="/files/ftp_upload/55029/55029fig4.jpg" /> Figura 4: Efecto de DMSO sobre la frecuencia cardíaca de abeja. El gráfico muestra el porcentaje de variación de la frecuencia cardíaca basal, tal como se mide en latidos por minuto. El tratamiento de vaso dorsal abeja de la miel con 0,1% o 1% de DMSO v / v en solución al cuarto de Ringer no aumenta de manera significativa o disminuir la frecuencia cardíaca, con relación a la solución de fuerza trimestre de Ringer sin DMSO. El tratamiento con 5% o 10% DMSO en solución provoca una disminución significativa en la frecuencia cardíaca. Los resultados se expresan como media ± SEM (n = 10). * P &lt;0,05 frente a control de DMSO del 0%<html> (Prueba de Kruskal-Wallis con la prueba de comparaciones múltiples de Dunn). <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55029/55029fig4large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> Para que este ensayo sea relevante, debe ser posible observar cambios en la frecuencia cardiaca tras el tratamiento con compuestos cardiomodulatory. La octopamina amina biogénica se sabe que actúa como un neuromodulador en invertebrados 10 y se ha demostrado que actúa como un cardioaccelerant tanto en moscas de la fruta 11 y abejas de miel 7. Octopamina se ensayó frente a vehículo (DMSO al 1% en solución al cuarto de Ringer) como se describe anteriormente a concentraciones de 100 nM, 10 mM, y 100 mM. Figura 5 muestra los resultados de este experimento, lo que demuestra que 10 mM y 100 mM concentraciones de octopamina aumentaron significativamente la frecuencia cardiaca. t &quot;fo: keep-together.within-page =&quot; 1 &quot;&gt; <img alt="Figura 5" src="/files/ftp_upload/55029/55029fig5.jpg" /> Figura 5: Efecto de la octopamina en el ritmo cardíaco de abeja. El gráfico muestra el porcentaje de variación de la frecuencia cardíaca basal, tal como se mide en latidos por minuto. El tratamiento de vaso dorsal abeja de la miel con 100 nM octopamina no aumenta significativamente o disminuir la frecuencia cardiaca con respecto al vehículo (1% de DMSO v / v en solución al cuarto de Ringer). El tratamiento con 10 mM y 100 mM octopamina aumentó significativamente la frecuencia cardiaca respecto al vehículo. Los resultados se expresan como media ± SEM (n = 10). * P &lt;0,05 frente al control del vehículo (prueba de Kruskal-Wallis con la prueba de comparaciones múltiples de Dunn). <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55029/55029fig5large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>

Watch this Scientific Journal Video about Dissection and Observation of Honey Bee Dorsal Vessel for Studies of Cardiac Function at JoVE.com

Dissection and Observation of Honey Bee Dorsal Vessel for Studies of Cardiac Function

The abdominal dorsal vessel of the honey bee and other insects serves as the functional equivalent of the mammalian heart and plays an important role in nutrient transport, waste removal, immune function, and more. Here we describe a protocol for the visualization and pharmacological manipulation of bee heart rate.

La disección y la observación de la abeja de la miel dorsal del recipiente de Estudios de la función cardiaca

The European honey bee, Apis mellifera L., is a valuable agricultural and commercial resource noted for producing honey and providing crop pollination ...

dissection-observation-honey-bee-dorsal-vessel-for-studies-cardiac

Nanyang Technological University

Research

JoVE Journal

Biology

10.8K vistas.  Virginia Tech. The abdominal dorsal vessel of the honey bee and other insects serves as the functional equivalent of the mammalian heart and plays an important role in nutrient transport, waste removal, immune function, and more. Here we describe a protocol for the visualization and pharmacological manipulation of bee heart rate.

Video: Dissection and Observation of Honey Bee Dorsal Vessel for Studies of Cardiac Function

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to measure odor evoked activity in the antennal lobe1. From then on, it has been continuously refined and applied to different neuropiles in the bee brain. Here, we describe the preparation currently used in the lab to measure activity in mushroom body neurons using a dextran coupled calcium-sensitive dye (Fura-2). We retrogradely stain mushroom body neurons by injecting dye into their axons or soma region. We focus on reducing the invasiveness, to achieve a preparation in which it is still possible to train the bee using PER conditioning. We are able to monitor and quantify the behavioral response by recording electro-myograms from the muscle which controls the PER (M17)2.
After the physiological experiment the imaged structures are investigated in greater detail using confocal scanning microscopy to address the identity of the neurons.

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

Bees can be conditioned in an appetitive olfactory learning paradigm (PER-conditioning). Using odors as stimuli, we established a method in which behavior is recorded while simultaneously Calcium Imaging is used to measure odor evoked activity in mushroom body neurons in vivo.

En vivo Ca 2 + - Imágenes de las neuronas del cuerpo de hongos durante el aprendizaje olfativo en la abeja de la miel

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to ...

Investigación

Biología

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

Monitorización de la función del corazón en las larvas Drosophila melanogaster Para los estudios fisiológicos

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagénesis y análisis funcional de los canales iónicos heteróloga se expresa en células de mamífero

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Básico<em&gt; Caenorhabditis elegans</em&gt; Métodos de sincronización y de observación

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

Microscopia intravital de la microcirculación en el músculo de ratón Cremaster para el análisis de la migración de la célula madre periféricas

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Obtaining Specimens with Slowed, Accelerated and Reversed Aging in the Honey Bee Model

Societies of highly social animals feature vast lifespan differences between closely related individuals. Among social insects, the honey bee is the best established model to study how plasticity in lifespan and aging is explained by social factors.
The worker caste of honey bees includes nurse bees, which tend the brood, and forager bees, which collect nectar and pollen. Previous work has shown that brain functions and flight performance senesce more rapidly in foragers than in nurses. However, brain functions can recover, when foragers revert back to nursing tasks. Such patterns of accelerated and reversed functional senescence are linked to changed metabolic resource levels, to alterations in protein abundance and to immune function. Vitellogenin, a yolk protein with adapted functions in hormonal control and cellular defense, may serve as a major regulatory element in a network that controls the different aging dynamics in workers.
Here we describe how the emergence of nurses and foragers can be monitored, and manipulated, including the reversal from typically short-lived foragers into longer-lived nurses. Our representative results show how individuals with similar chronological age differentiate into foragers and nurse bees under experimental conditions. We exemplify how behavioral reversal from foragers back to nurses can be validated. Last, we show how different cellular senescence can be assessed by measuring the accumulation of lipofuscin, a universal biomarker of senescence.
For studying mechanisms that may link social influences and aging plasticity, this protocol provides a standardized tool set to acquire relevant sample material, and to improve data comparability among future studies.

In honey bee workers, aging depends on social behaviors rather than on chronological age. Here we show how worker-types with very different aging patterns can be obtained and analyzed for cellular senescence.

La obtención de especímenes con desacelerado, acelerado y Invertida Envejecimiento en la abeja de la miel Modelo

Societies of highly social animals feature vast lifespan differences between closely related individuals. Among social insects, the honey bee is the best ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

Reconstitución funcional y la actividad del canal Las mediciones de Purificada Wildtype y mutante CFTR Proteína

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Methods for Comparing Nutrients in Beebread Made by Africanized and European Honey Bees and the Effects on Hemolymph Protein Titers

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees collect and convert to beebread by placing colonies in a specially constructed enclosed flight area. Methods were developed to analyze the protein and amino acid composition of the pollen and beebread. We also describe how consumption of the beebread was measured and methods used to determine adult worker bee hemolymph protein titers after feeding on beebread for 4, 7 and 11 days after emergence. Methods were applied to determine if genotype affects the conversion of pollen to beebread and the rate that bees consume and acquire protein from it. Two subspecies (European and Africanized honey bees; EHB and AHB respectively) were provided with the same pollen source. Based on the developed methods, beebread made by both subspecies had lower protein concentrations and pH values than the pollen. In general, amino acid concentrations in beebread made by either EHB or AHB were similar and occurred at higher levels in beebread than in pollen. Both AHB and EHB consumed significantly more of the beebread made by AHB than by EHB. Though EHB and AHB consumed similar amounts of each type of beebread, hemolymph protein concentrations in AHB were higher than in EHB. Differences in protein acquisition between AHB and EHB might reflect environmental adaptations related to the geographic region where each subspecies evolved. These differences could contribute to the successful establishment of AHB populations in the New World because of the effects on brood rearing and colony growth.

Here are methods to quantify nutrients in pollen before and after its conversion to beebread by two subspecies of honeybees. We describe techniques to measure beebread consumption and resulting protein titers in both subspecies.

Los métodos para la comparación de Nutrientes en jalea real Realizado por africanizadas y europeas Honey Bees y los efectos sobre títulos hemolinfa de proteínas

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

Análisis de la transferencia dependiente de la unión Gap de miARN con 3D-FRAP Microscopía

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

Monitoring Colony-level Effects of Sublethal Pesticide Exposure on Honey Bees

The effects of sublethal pesticide exposure to honey bee colonies may be significant but difficult to detect in the field using standard visual assessment methods. Here we describe methods to measure the quantities of adult bees, brood, and food resources by weighing hives and hive parts, by photographing frames, and by installing hives on scales and with internal sensors. Data from these periodic evaluations are then combined with running average and daily detrended data on hive weight and internal hive temperature. The resulting datasets have been used to detect colony-level effects of imidacloprid applied in a sugar syrup as low as 5 parts per billion. The methods are objective, require little training, and provide permanent records in the form of sensor output and photographs.

Sublethal doses of pesticides may affect colonies in ways that are difficult to detect using only periodic or visual methods. Methods to measure total adult bee mass, brood, and food resources by weighing hives and hive parts, photographing frames, and installing sensors, are provided. Data analysis is also addressed.

Controles Colonia efectos de exposición a los pesticidas subletales en las abejas de miel

The effects of sublethal pesticide exposure to honey bee colonies may be significant but difficult to detect in the field using standard visual assessment ...