LABORATORY INQUIRY Effects of Pesticides on Arthropod Nervous Systems

by Dana Krempels, Ph.D. Along with the muscular system, the nervous system is unique to animals. This complex system of interacting cells allows animals to react to both internal and external stimuli. Animals also interact with each other, and most species are involved in one or more forms of symbiosis. One example of symbiosis is parasitism, in which a parasite benefits at the expense of a host, usually by feeding on the host without killing it outright. An example of parasitism familiar to just about anyone who has ever had a mammal as a pet is the relationship between a host mammal and different types of invertebrate parasites such as fleas, ticks, mites, and roundworms.

In recent years, several drugs have been developed that kill various species of arthropods and nematodes without apparent harm to the host mammal. That a single product can kill both arthropods and nematodes might come as a surprise to someone who looks only at the superficial dissimilarity of the two types of animals (Figure 1). However, Arthropoda and Nematoda share a relatively recent common ancestry (Dopazo & Dopazo 2005), and are both classified in the taxon Ecdysozoa (the “moulting animals”). Because of their common ancestry, they may also share some physiological characteristics inherited from their common ancestor.

Figure 1. A sampling of Ecdysozoans. a – Caenorhabditis elegans, a free-living nematode; b – Strongyloides stercoralis, a nematode parasite that inhabits the intestines or lungs of mammals; c – Drosophila melanogaster, the Black-bellied Fruit Fly; d – Dermacentor sp., the Rocky Mountain Spotted Fever Tick; e – Ornithonyssus bacoti, the Tropical Rat Mite; f - Ctenocephalides canis, the Dog Flea; g - Lucilia sericata, a Green Bottle Blowfly. The evolutionary relationships of these animals can be viewed on a phylogenetic tree at http://www.tolweb.org/Arthropoda/2469

Pharmaceutical companies have made good use of this evolutionary relationship. Drugs such as Revolution (selamectin), Frontline (fipronil), Advantage (imidocloprid), and Capstar (nitenpyram) can be used to kill both arthropod and nematode parasites, though their efficacy against each type of parasite can vary. They kill parasites ostensibly without harming the mammalian host.

The U.S. Food and Drug Administration (FDA) has approved the use of the aforementioned drugs on dogs and cats only. However, some veterinarians use the drugs “off label” (see:

http://en.wikipedia.org/wiki/Off-label_use) to treat parasites in other species, from hoofed livestock to rodents and lagomorphs. Is this use warranted? Safe? Effective?

In this lab, you will have the opportunity to learn how these drugs act—at the cellular and molecular level--on the nervous system of the parasites without harming the host (or do they?), and to design an experiment to explore their efficacy at killing the unwanted parasite, whether used as directed or off label. The key is to understand the animal nervous system, its working components, and how these operate and differ between parasite and host. I. The Nervous System: An Overview

One characteristic that separates animals from all other life forms is the presence of a nervous system. This complex organ system is composed of specialized cells known as neurons (Figure 2), which can be various types, depending on function and location. Neurons coordinate an animal’s actions, allowing it to respond appropriately to internal and external stimuli.

You may be most familiar with the vertebrate nervous system, which consists of two parts, the central and peripheral nervous systems. The central nervous system consists of the brain and spinal cord, whereas the peripheral nervous system includes the sensory neurons, neuron clusters (ganglia), and the nerves connecting the sensory neurons and ganglia to the central nervous system. Not all animals have a nervous system as complex as that of vertebrates, and many have little more than a cluster of neurons (known as a ganglion) for a “brain”.

Figure 2. Schematic diagram of a generalized vertebrate neuron. The myelin sheath is unique to vertebrates, and facilitates more rapid signal transmission.

When a neuron is adequately stimulated, its membrane potential rises and falls rapidly, beginning at the axon hillock and traveling towards the axon terminals. This action potential is central to communication between neurons. The action potential is transmitted from one neuron to the next via the action of chemical compounds, produced by the neurons themselves, called neurotransmitters. If the reception of a neurotransmitter results in propagation of the action potential, excitation has occurred. Conversely, if the reception of a neurotransmitter stops the action potential from propagating, inhibition has occurred.

A. Neurotransmitters A neurotransmitter is a chemical produced by a neuron that transmits an action potential from one neuron (the presynaptic cell) to the next neuron (the post-synaptic cell) across a microscopic gap, the synapse, between the presynaptic axon terminals and receptors on the postsynaptic dendrites (Figure 3).

Although neurotransmitters are often labeled “excitatory” or “inhibitory”, it is actually the nature of the receptor that causes an nervous signal to be transmitted (excited) or halted (inhibited). It is true that certain neurotransmitters always (or almost always) bind to receptors that generate an electrochemical signal that travels along the neuron’s axon (action potential). Most neurotransmitters (e.g., glutamate) fall into this category. However, the major receptors (most, but not all) of certain other important neurotransmitters, such as Gamma aminobutyric acid (GABA) are inhibitory. Still other neurotransmitters (e.g., acetylcholine) may elicit excitation or inhibition, depending on the receptor to which they bind.

Figure 3. A generalized synapse. Neurotransmitters from the presynaptic neuron cross the synapse and bind to receptors on the postsynaptic neuron. This results either in propagation of the action potential (excitation) or non-propagation of the action potential (inhibition), depending on the nature of the receptor to which the neurotransmitter binds. (After http://en.wikipedia.org/w/index.php?title=File:Synapse_Illustration_unlabeled.svg&page=1) B. Neurotransmitter Receptors Neurons communicate with each other and with other cells via electrochemical waves that travel along their axons. In vertebrates, the axons are encased in myelin, which causes the signal to travel more quickly than in a non-myelinated axon (such as those found in non-vertebrates). The gap between the axon terminals of one neuron and the dendrites of the next neuron is known as a synapse. Once the electrochemical signal of a pre-synaptic neuron reaches the axon terminals, the gap must be bridged by neurotransmitters. These are released from presynaptic vesicles, and travel across the synapse to neurotransmitter receptors (a.k.a. neuroreceptors) on the dendrites of the postsynaptic neuron.

Receptors on the membrane of a postsynaptic neuron receive neurotransmitters, and generate an electrical signal by regulating the activity of ion channels. Binding of particular neurotransmitters to specific neuroreceptors can change membrane potential, generating an action potential that travels along the axon of the postsynaptic cell until it reaches the axon terminals (at which point the postsynaptic cell becomes the presynaptic cell, relative to the next synapse), and the process repeats itself, serially, along a line of neurons.

Animals have two types of receptors, ionotropic receptors (a.k.a. ligand-gated receptors) and metabotropic receptors (a.k.a. G protein-coupled receptors). Ligand-gated receptors can be either excited (i.e., made more likely to generate an action potential) or inhibited (i.e., made less likely to generate an action potential), depending on the type of neurotransmitter received.

One might make an (admittedly imperfect) analogy of excitatory signals being like the gas pedal of the nervous system, allowing movement to proceed, and the inhibitory signals being like the brakes, stopping the transmission.

Unlike ligand-gated receptors, G protein-coupled receptors modulate the actions of excitatory and inhibitory neurotransmitters, and are neither excitatory or inhibitory. Most animal receptors are G protein coupled, and this type of receptor is extremely important in modern medicine, both human and veterinary.

When received by a ligand-gated receptor, neurotransmitters such as dopamine, glutamate and epinephrine are excitatory. Others, such as Gamma aminobutyric acid (GABA) and glycine, are usually inhibitory.

II. Parasites and Hosts: Weaponizing Neurotransmission

Not all neurotransmitters, receptors, and neurons have evolved to be the same in the many animal lineages. Significant differences in the physiology between Ecdysozoans and Vertebrates can be used to wage war against the Ecdysozoan parasites that plague our companion animals and agricultural livestock. Several drugs used in veterinary medicine interfere with the normal function of neurotransmitters and receptors in their target parasites, while (hopefully) not causing problems in the host’s own nervous system. There are several different classes of these drugs, some of which are briefly described below. As you will discover, the exact mechanism of action is not always completely understood.

A. Avermectins There are several different avermectins, but the most commonly used in veterinary medicine are ivermectin (used to treat internal and external parasitic ecdysozoans in hoofed livestock), and selamectin (Brand name Revolution or Stronghold; approved for use in dogs and cats to control internal nematodes and external arthropod parasites). While the exact mechanism of the avermectins is still under study, it is believed that they bind to glutamate-gated chloride channels in the ecdysozoan nervous system. This increases the membrane channels’ permeability to chloride ions, inhibiting transmission of action potentials and resulting in paralysis and death. Ivermectin is injected or given orally. Selamectin is applied topically, spreads rapidly across the oil on the skin and the hair follicles. The drug is also absorbed through the skin and enters the bloodstream, where it is delivered to internal tissues.

B. Chloronicotinyls Chloronicotinyls are synthetically derived relatives of natural nicotine, which is produced by certain plants to deter herbivory by insects. Veterinarians currently can use two compounds in this class for treating parasites in livestock and companion animals, imidocloprid (brand name, Advantage) and nitenpyram (brand name, Capstar).

Imidacloprid (Brand name, Advantage) is a chloronicotinyl insecticide, a synthetically chlorinated derivative of nicotine. Drugs in this class bind to nicotinic acetylcholine receptors in the arthropod central nervous system (brain and ventral nerve cord). This causes inhibition of cholinergic action potential transmission, resulting in paralysis and death.

Imidacloprid is applied topically to control fleas and lice. Nitenpyram is administered by mouth (pill form) to kill fleas. One of the selling points of Capstar is its rapid activity: within 20 - 30 minutes of administration, fleas begin to drop off their hosts and die. Within four hours, all fleas are dead. Unliike imidocloprid, nitenpyram promotes hyperexcitability in nicotinic neuron receptors, resulting in death.

C. Phenylpyrazoles The most commonly used phenylpyrazole is fipronil (brand name, Frontline), used to control both nematode and arthropod parasites. The drug binds GABA receptors in insects, inhibiting influx of chloride ions into nerve cells. This causes hyperexcitability of the neuron, resulting in death. Fipronil is applied topically to the skin. It is absorbed into the bloodstream, and also accumulates in the oil glands on the skin. Because it is not very soluble in water, it remains on the animal for a longer time than some of the other parasiticides, and has a long “hang time.” (This can be problematic in mammal species in which fipronil is neurotoxic, such as rabbits and some carnivores. Once Frontline is applied, little can be done to save an animal suffering the neurotoxic effects.)

D. Other Parasiticides The above are some of the most commonly used parasiticides in veterinary medicine, but there are many others. For a more complete overview, the Merck Veterinary Manual is a good source. More information can be found here:

http://www.merckvetmanual.com/mvm/index.jsp?cfile=htm/bc/191907.htm

http://www.merckvetmanual.com/mvm/index.jsp?cfile=htm/bc/191903.htm

http://www.merckvetmanual.com/mvm/index.jsp?cfile=htm/bc/190504.htm III. Roundworms, and Maggots, and Fleas, Oh my!

The drugs described in the preceding section are almost all used primarily to kill fleas. Some of them have been anecdotally reported to be effective against nematodes, but controlled studies are often lacking to support this claim. Some of these drugs also are effective against lice, but others are not. Similarly, some of these drugs will kill mites and some ticks, but not all of species.

A. Myiasis The Order Diptera is a very large and diverse taxonomic group within the Ecdysozoa. It includes the insects we affectionately call flies. House flies, Fruit flies, Blow flies, and mosquitoes are all members of this group, and all share a common ancestor from which they inherited two flying wings (hence, the name of the taxon, which literally translates as “two wings”) and two modified hindwings called halteres, used as gyroscopes to help the animal balance and maneuver in flight.

Flies share more than their superficial morphology, however. They all have a similar life cycle, hatching from eggs to become maggots, which feed for a juvenile growth period, then pupate. The pupa develops in its cocoon and finally hatches out as an adult fly.

Some flies are cute and nice (fruit flies), some are annoying (house flies), and some can be deadly (blow flies). Some species of flies make their living by laying their eggs on dead and decaying flesh. Maggots perform a valuable ecological function by eating carrion and reducing dead, organic matter to smaller particles that can then be fully decomposed by bacteria and fungi. But sometimes things go terribly wrong, at least if you are the victim of “fly strike,” technically known as myiasis (Figure 4). Myiasis is the infestation of a living host’s tissue with a parasitic fly larva. It can be the larvae of a botfly (truly disgusting, but not usually fatal) or a blowfly (possibly even more disgusting (it’s a judgment call), but potentially fatal). If you’re not familiar with myasis, YouTube will be happy to help you learn more. But keep that barf bag handy. Flies use chemoreception (“smell”) to locate food and suitable places to lay their eggs. Evolution has provided them with a broad spectrum of sensitivity, and they can be enticed by a variety of smells, from that of fecal matter to that of wet fur that has accumulated a population of smelly bacteria. When these smells are emanating from a bird or mammal, adult blowflies may lay their eggs on the animal. When the maggots hatch out, they begin to consume, first the tainted fur, and then the living flesh of the host. As they feed, they cause more damage with their digestive enzymes, and this attracts more flies. The result can be devastating and fatal in a relatively short time, as fly eggs can hatch in less than 24 hours. (See http://www.nhm.ac.uk/research-curation/research/projects/myiasis-flies/ for more information on how myasis-causing flies find their hosts.)

It is thus of great veterinary interest to find drugs that can quickly kill maggots infesting living flesh, as they sometimes create cavities that are difficult to reach, even with tweezers and other surgical tools. If you do a Google image search with keyword “myasis”, you may never sleep again.

IV. Designing a Project

For this lab project, your team should study the introduction above carefully, and consider what question/problem to address regarding the effect of various veterinary parasiticides on a select model organism (or organisms). The following section outlines a sample project in which the effect of selamectin on fly larvae is investigated. Your team should use this type of approach to address a similar question. The descriptions of the evolutionary relationships of the parasites, the mode of action of the parasiticides, and other information in the previous sections may give you some ideas for a project. Note that you should include similar, relevant background information in your presentation. This

should include why you chose a particular model organism, why your question is important/relevant, and the possible applications of your findings. A. Sample Project: The Effect of Selamectin on Green Blowfly Larvae (Lucilia sericata)

Introduction: Blowflies and the Economic Importance of Myiasis Blow flies (also known as carrion flies, bluebottles, or greenbottles) are insects (Order Diptera) that provide the important ecological function of helping to break down dead, organic matter into smaller components that can be decomposed by bacteria and fungi. These flies are attracted to carrion (dead animals), garbage, compost, and other rich sources of energy that have a strong smell. Here, they lay their eggs and larvae hatch out within 24 hours to begin feeding and growing. Depending on temperature and other environmental conditions, the larvae can pupate within 5-8 days, and then emerge as adults in another few days.

Unfortunately, blow flies are sometimes attracted to strong smells on living animals (including humans) who have wounds, fecal matter on their fur, or are compromised in health and exuding a smell that attracts the flies. When the flies lay eggs on a living animal, their larvae can do serious damage in a relatively short time. The presence of maggots on a host may attract additional adult flies to the site, compounding the problem.

An animal discovered to be suffering from “fly strike” (myiasis) must have the maggots physically removed or killed with medication to prevent life-threatening harm. It is important to use medication that will target the parasite’s physiology without harming to the host. Because the nervous systems of vertebrates and ecdysozoans differ in many respects, pharmaceutical companies have developed drugs that target ecdysozoan systems preferentially. One of these is an avermectin-class drug, ivermectin.

Ivermectin acts as a GABA agonist (i.e., it binds to GABA receptors, thus mimicking and increasing the effects of the neurotransmitter GABA), causing paralysis in certain arthropods and nematodes. The drug is approved for use in dogs and cats to combat the mite species Sarcoptes scabeii , Otodectes cynotis , Cheyletiella blakei , C yasguri , and Demodex canis . It is approved for use in cattle for to treat mange caused by Psoroptes mites, as sell as lice psoroptic mange, lice, and larvae of warble flies. In horses and humans, it is used to treat nematode infections of the skin and intestine.

In mammals, GABA is found only in the central nervous system (CNS). Because ivermectin does not readily cross the blood-brain barrier, it will kill parasites at about 1/10 the dose that would elicit a toxic reaction in a mammalian host. (Oddly, some dog breeds (Collies, Shetland Sheepdogs, Old English Sheepdogs, Australian Collies, and their hybrids) are sensitive to ivermectin, as it appears to penetrate the blood/brain barrier in these breeds. Relatively small doses can produce transient neurological signs, but at higher doses, it can be fatal.)

Flies (and their larvae) are ecdysozoans that share a common ancestor with the nematode parasites known to be susceptible to ivermection. If fly larvae share the nervous system characteristics that make nematodes susceptible to ivermectin, it is possible that ivermectin could be used to treat myiasis.

Hypothesis and Predictions The working hypothesis for this investigation is:

Larvae of the Green Blowfly (Lucilia sericata) share nervous system characteristics with nematodes and arachnid ecdysozoans that make them susceptible to the GABA-agonist action of ivermectin.

Considering these factors, we can make the following prediction to test the hypothesis.

If the larval nervous system is disrupted by ivermectin, larvae will suffer more adverse effects when given ivermectin than when given a control substance/placebo.

To test the hypothesis, we will subject two populations of Lucilla sericata larvae to different treatments, one being treated ivemectin, and the other being treated with the inactive carrier commercially used to dissolve ivermectin. The null and alternative hypotheses are:

HO – There will be no difference in mobility/survival in Lucilla sericata larvae treated with ivermectin versus those treated with an inert carrier susbstance (95% ethanol). HA - There will be a difference in mobility/survival in Lucilla sericata larvae treated with ivermectin versus those treated with an inert carrier susbstance (95% ethanol).

Methods: Disrupting Nervous System Function in a Dipteran Parasite Ivermectin is used to treat nematode infections in the skin, eyes, intestines, and other tissues and organ systems. The dose typically prescribed for a mammal is 0.2mg/kg of body weight. Dosage used to treat these parasites depends on the parasite and the species of the host, and can range from 0.3 – 0.6mg/kg given orally or injected subcutaneously for one treatment, and then repeated 10-14 later to kill any newly hatched larvae.

Materials 10mg ivermectin 2ml 95% ethanol 30 glass culture tubes containing prepared, commercial growth medium 10 culture jars gauze sheeting rubber bands paper toweling small (art) paintbrushes (for handling larvae) millimeter ruler 4 glass petri dishes for observations 4 filter paper disks water-based marking pens compound stereoscope for observations Procedure Ivermectin is a crystalline powder insoluble in water, but soluble in 95% ethanol. We will make a 10mg/ml solution of ivermectin by dissolving 10mg of ivermectin in 95% ethanol to a total volume of 1 milliliter. This will be applied to the surface of larval growth medium at a dose 0.2mg/kg of culture growth medium. The fly culture set-up is illustrated in Figure 4.

Because it is important to distinguish the effects of ivermectin from those of its carrier, 10 cultures of fly larvae (10 larvae per culture) will be treated with ivermectin dissolved in 95% ethanol, and 10 cultures of larvae (10 larvae per culture) will be treated with the same volume of 95% ethanol alone.

Larvae will be allowed to move and feed for one hour. At specific, noted time intervals, larvae from each treatment and each control culture will be placed, one at a time, on a petri dish containing a filter paper disk, and their rate of movement measured (millimeters per ten second trial). As the larva moves across the paper, a small, colored dot will be made (with a water-based marking pen) one millimeter behind the organism’s tail, so as to avoid disturbing it or inadvertently increasing its movement. After 30 seconds, the trial will end, and the length of the larva’s path will be measured. (If the path is not straight, a string will

be laid along the path and its length measured.) The data will be converted to a rate (mm/min).

Figure 4. Culture set-up for both treatment and control fly larvae. For multiple cultures, a jar such as that shown in (A) may be employed. For single cultures, a cotton plug will be used to prevent contamination (B), and the tubes placed in a test tube rack. If desired, a heating pad can be placed under the jars with space between the pad and the jar to prevent overheating. A thermometer placed in the jar (through the gauze) will allow temperature to be monitored for control.

This procedure will be repeated for each larva, with the exact time post-treatment noted.

At 24 hours post treatment, all samples will be examined and the number of dead and alive larvae in each culture recorded.

At the end of all trials, the movement rate of the treatment and control groups will be calculated and plotted against time after initial treatment (with ivermectin or plain 95% ethanol). A student’s t test will be used to determine whether the average rate of movement in the treatment group at each time interval is significantly different from the average rate of movement in the control group at the same time interval.

Total mortality in both treatment and control groups will be measured, and a Chi-square test performed to determine whether there is a significant difference in mortality between treatment and control. Results and Analysis Use these ideas and apply them to your team’s research project.

1. At what stage of development were the larvae you used? Why is this relevant?

2. How long did it take for the drug to have an effect on larval locomotion?

3. How long did it take for the drug to kill the larvae, if they were killed?

4. Were all larvae equally affected by the drug in the treatment group? If not, what might this imply?

5. Do your data indicate rejection or failure to reject your competing hypotheses?

6. If ivermectin inhibits fly larvae from moving and surviving, what applications might this have? Explain in detail.

Follow-up Experiment If you did find a significant difference in rate of movement between treatment and control groups, the next step might be to determine the most effective dose of the drug to use in treating myasis caused by these larvae. LD-50 stands for Lethal Dose 50. This is the dose of a substance that causes 50% of the individuals in an experimental trial to die from the effects of the substance.

As a followup to your experiment on the effects of ivermection on larval movement and, your team might modify the dose of ivermectin to determine whether a higher dose results in greater/faster mortality, as well as the dose that leaves survivors behind to breed. B. Your Team’s Project Armed with the information in this chapter, your team should discuss possible projects to test the efficacy of selected parasiticides against the parasite of your choice. Model organisms that we can readily provide are the larvae (maggots) of both fruit flies and—if you really want a disgusting culture experience—blowflies. If you would like to work with nematodes, we can get those, too. But we need to know with enough advance time to place an order.

After reading through this lab chapter, and early in the lab period , your team will develop an overall hypothesis, prediction(s), and working (null and alternative) hypotheses. BEFORE YOU GO ANY FURTHER, your lab instructor must discuss your hypotheses and predictions with you to be sure they are logical and feasible to test. Once your instructor has approved your initial ideas, you must create a very thorough and complete experimental protocol. It should include all materials and items you will need (including exact quantities of everything) as well as the experimental steps you will perform. YOUR PROTOCOL SHOULD BE SO COMPLETE AND DETAILED THAT YOU COULD HAND IT TO ANOTHER TEAM AND THEY COULD PERFORM YOUR EXPERIMENT WITHOUT HAVING TO ASK YOU ANY QUESTIONS ABOUT HOW TO DO IT.

Figure 4. Examples of myasis, and why your project could be important. a – myiasis in tissue of a cat; b – myiasis in the infected root bed of a human tooth; c – myasis secondary to ocular neoplasia (cancer) in a cow; d – myasis in a Purple Martin nestling.

Once your protocol is ready, keep a copy for yourselves (one for each team member: everyone is responsible for knowing everything about the project!) and turn in a copy to your instructor. This must be given NO LATER THAN THE END OF THE LAB SESSION, as the lab staff needs the information to be able to order your supplies in a timely fashion.

Your team’s job now is to spend some time doing further research to: 1. Understand different types of neurotransmitters and neurotransmitter receptors in your model organism of choice. 2. Know your vocabulary: Be sure you know the meaning/significance of at least these terms: central nervous system peripheral nervous system neurotransmitter neuron (and all its anatomical components) receptor ligand neurotransmitter agonist neurotransmitter antagonist nicotinic receptor ligand-gated ion channel (ionotropic receptor) G protein-coupled receptors (metabotropic receptors) If your’e not sure—look it up!

3. Devise an overall hypothesis and competing experimental hypotheses. Make predictions, and design a rigorous, interesting experiment that could be of real, veterinary relevance.

4. Determine the materials you will need, and the exact quantities of each material or piece of equipment This means you will need to research the doses of these drugs used by veterinarians for treating various parasites. (If you get stuck, you may ask Dr. Krempels for help. But do this only as a last resort. YOU are the investigators here, and you need to use all the information technology resources at your fingertips!)

5. Report to your lab instructor the materials you will need NO LATER THAN THE END OF YOUR PLANNING SESSION IN LAB.

Depending on which model organism you choose, different culture methods will be necessary. You should research these on your own to determine whether raising these ecdysozoans in lab will be feasible and conducive to good experimentation.

Your lab instructor and the laboratory staff will be happy to work with your group to provide the materials you need to culture your creepy organisms and then…try to kill them.

You know your chemistry lab isn’t this fun, don’t you? Literature cited Dopazo, H, Dopazo J: Genome-scale evidence of the nematode-arthropod clade. Genome Biology 2005, 6:R41 (http://genomebiology.com/2005/6/5/R41)