Laboratory 7: Arthropod Vectors Arthropods are a highly successful group of animals unrivalled by any others in diversity of structure and function. They occur practically everywhere and have adapted to nearly all-imaginable sources of food, including vertebrate blood. Blood-feeding has arisen independently in several groups of arthropods and is based on the simple need for protein. This protein can be used for insect development (by immature stages) or for egg development (females). The transmission of parasites by arthropods is a result of this need for protein.

The arthropods you will examine today represent two Classes of Arthropods: Insecta and Arachnida. Many of the arthropods you learn about in this lab are the vectors for parasites we’ve discussed in previous lab sessions. Learn to place the vector with the parasite!

Class Insecta: Insects also show huge variety in shape and form. Almost the only condition their group does not attain is very large body size. A number of features, however, are shared by most kinds of living insects: a head, thorax, and abdomen; a pair of relatively large compound eyes, a pair of antennae, mouthparts consisting of a labrum, a pair of mandibles, a pair of maxillae, a labium, two pairs of wings, and three pairs of walking legs.

Order Diptera The Diptera are one of the largest insect orders with over 100,000 species identified in 140 families. They can vector many disease agents.

• Tse-tse files (examine the slide of Glossina) can transmit the agent causing African sleeping sickness (Trypanosoma spp).

• Mosquitoes can transmit Plasmodium spp, viruses, and filarial nematodes. • Biting midges transmit filarial nematodes. • Tabanid flies transmit tularaemia.

Suborder Nematocera Family Culicidae: Mosquitoes Life cycle: Mosquitoes require water for the development of larvae and pupae. Eggs are laid in or near water. They have 4 larval stages (which can last anywhere from 3 days to over 1 month). The fourth larval stage is the last stage before the pupal stage. The pupal stage is a free-swimming but non-feeding stage of development. Adults will emerge from the pupae at the surface of the water and mate. Females will seek out a host and take a blood meal. She then moves on to lay her eggs.

Females are attracted to a host by several cues such as CO2 and odour. Once a host is located, the female will probe for capillaries. Once a capillary is found, the cibarial pump is activated and then midgut is filled. Stretch receptors in the midgut of the mosquito respond in order to stop feeding.

Most mosquitoes have preferences for certain groups of vertebrate hosts; but will switch hosts if the situation demands. This results in the transfer of disease agents between different groups of hosts, as is the case with the transmission of West Nile virus from birds to humans.

Aedes aegypti This species is found mainly in the tropics and sub-tropics, and is known as the yellow fever mosquito. Aedes aegypti transmit several medically important human arboviruses including Yellow fever virus, Dengue virus, and in some cases West Nile viruses. Mosquitoes in the genus

Aedes are of medical importance because they have many behavioural characteristics that make them important vectors of the parasites that cause human and animal diseases. They

• are container breeders because they will lay their eggs in tin cans, flower pots or any other small volume of water and so are hard to eradicate as larvae.

• live in or near human dwellings and typically fly only a few hundred meters from their site of emergence.

• feed on humans as preferred hosts

Pinned specimen: Aedes aegypti

Family Psychodidae – Sandflies Adult sand flies are small (~ 2mm) and may be recognized by their hairy bodies and wings that are held erect over their body. Their small size makes them difficult to study.

Sand flies transmit both protistan parasites and viruses. Leishmania, the causative agent of leishmaniasis, is transmitted by sand flies. Sand flies are also vectors for a Phlebovirus (causing sand fly fever) and a vesiculovirus (causing vesicular stomatis in humans and animals).

Pinned specimen: Sandflies Family Simuliidae – Black flies Black flies are associated with well-aerated aquatic habitats. Because the females are aggressive feeders, black flies transmit several diseases to humans. The disease agents transmitted by black flies include viruses, apicomplexans, trypanosomes, and filaroid nematodes. The most important of these disease agents from a human perspective is Onchocercus volvulus. Review your notes on the life cycle and development of Onchocercus volvulus.

Pinned specimen: Black fly

Family Tabanidae Horse flies and deer flies may transmit the bacterial pathogens of tularemia (Pasteurella tularensis) and anthrax (Bacillus anthracis), as well as a parasitic roundworm (Loa loa) that causes loiasis in tropical Africa.

Pinned specimen: horse fly Order Hemiptera: Hemipterans, or true bugs, comprise one of the largest orders of insects with more than 55,000 species described world-wide. Relatively few are ectoparasites of mammals and birds. Hemipterans are exopterygotes (wings develop externally) with hemimetabolous development (the nymphs have habitat and feeding preferences similar to the adults). They all have piercing-sucking mouthparts well-adapted for feeding on plants, small arthropods and vertebrate blood. Two families are of medical importance: Cimicidae (bed bugs) and Reduvidae (assassin bugs).

Family Reduvidae: subfamily Triatominae Most reduvids are predators on other insects, thus the name “assassin bugs”. Most of these can, but usually do not bite humans. One subfamily within the Reduvidae, the Triatominae, is of medical important because they server as vectors for the trypanosome parasite, Trypanosoma cruzi, the causative agent of Chagas disease. Some important species in this subfamily include: Triatoma dimindiata, Rhodnius prolixus and Triatoma infestans. Triatomes are found in the warmer climates of the western hemisphere including the US and 3/5 the length of South America. Triatomes are usually associated with burrowing animals, such as rodents, squirrels and armadillos, or with nesting animals such as lizards and birds. Those that have become domiciliated will feed on dogs, humans and farm animals.

Rhodnius prolixus The triatome life cycle consists of an egg (development time~1 month) and 5 nymphal instars (involving a series of feedings and moltings). For R. prolixus, it generally takes 80 days of development time from egg to adult. Eggs are laid off the host in burrows, cracks or crevices, or thatched roofs for those insects that are domiciliated. In human settings, R. prolixus will hide in the walls or roof during the day. The number of bugs found in houses is directly related with the quality of housing. R. prolixus, like the other Triatomes, is attracted to warmth, CO2, and odours. Feeding takes place primarily at night. Triatome insects are capillary feeders. They have mandibles that are used to hold onto the skin and maxillae that probe the skin until a capillary is found. Feeding can take anywhere from 3-30 minutes. They will often feed on areas of the face where skin is thin (lips and eyes) but will also feed on other areas of the body. Over a lifetime, a triatome insect can take between 4 and 10ml of vertebrate blood!

R. prolixus is just one of the triatomes known to vector Trypanosoma cruzi. Review your notes on the development of T. cruzi in the vector and in the human host

Pinned specimen: R. prolixus

Family Cimicidae: bed bugs There are approximately seven species of bedbugs that will feed on humans. The most common bedbugs are Cimex lectularius (the "human bedbug"), Cimex hemipterus (the "tropical bedbug"), and Leptocimex boueti (found in W. Africa). Even though bedbugs have many characteristics that would make them excellent vectors for diseases, bedbugs do not, as far as is known, serve as vectors for any human diseases. Bedbugs are small (about 8 mm long), nocturnal creatures, coming out of hiding at night to feed on unsuspecting humans. They feed exclusively on blood! Their bites often result in an allergic reaction. Some humans tend to react more strongly to bedbug bites while others may never react no matter how many times they are bitten. In those instances where infestations are heavy and people are bitten many times, the bites are so annoying that a person will lose considerable sleep, and they may suffer from iron and/or hemoglobin deficiencies due to the lose of blood.

Pinned and preserved specimens: Cimex lectularius Order Siphonaptera: Fleas Fleas are highly specialized insects well adapted for a symbiotic life. All fleas consume blood as adults; using birds and mammals as their hosts. Most fleas exhibit low host specificity, though some do show high host specificity. General flea life cycle includes an egg, three larval instars, pupae, and an adult stage. Under optimal conditions, the life cycle can be completed within 2 weeks, but can be up to 2 years under suboptimal conditions. The crucial stage to long term survival is the pupal stage. Fleas are very sensitive to the presence of a potential host. They are sensitive to vibration, CO2, heat and direct pressure. Fleas will remain in the cocoon until when stimulated. Following stimulation, they emerge quickly and leap to reach a potential host.

Many characteristics make fleas successful parasites and vectors of disease. Think about what those characteristics might be.

Xenopsylla sp, the rat flea, is a vector of plague. Slide: Xenopsylla male and female Note the spermatheca of females and the copulatory organ of males that make recognition of sexes simple in this group. The genus Xenopsylla carries the bacterium, Yersinia pestis, which caused the Bubonic Plague and killed 25 million people during the last pandemic in the 14th century.

Yersinia pestis cycles in rodent populations in the wild or in urban areas. Fleas become infected when feeding on rodents during the bacteremia phase. The bacteria will multiply in the intestines of the fleas. Transmission from the flea back to another rodent occurs through “blocked” fleas. In blocked fleas, the bacteria multiply to high numbers and move into the anterior portion of the intestinal tract and block the proventriculus. The bacteria make a sticky mass, or plug that prevents the esophogeal pump from taking in blood. The flea attempts to take blood, fails to do so, when it relaxes, ejects bacteria back into the wound. Transmission also occurs through fecal droplets contaminated with the bacilli.

Order Mallophaga and Anoplura: the Lice: Lice are hemimetabolous insects that are often just a nuisance but can also transmit important diseases. They are wingless, small (< 5 mm), dorsoventrally flattened and have many short bristles or setae. They have large claws that they use to remain attached to the host. Lice are commonly divided into two separate orders: Mallophaga (chewing lice, or bird lice) and Anoplura (sucking lice).

The lice life cycle consists of an egg, 3 nymphal instars, and an adult. The adult stage is reached within 2-3 weeks. Body lice spend most of their time in clothing and will oviposit there rather than on the body. They cannot survive long off the host and can only survive unfed for about 10 days. Transmission from one host to the next is by close contact.

The Mallophaga have broad heads and strongly sclerotized mandibles because chewing mouthparts demand more power. They are most commonly parasites of birds. Mallophaga feed on bits of skin and feather debris, as well as skin secretions. A few feed on host blood.

The Anoplura have elongated heads with the sucking mouthparts protruded from the anterior tip of the head. To feed they cut through the host’s skin with their teeth, and probe with stylets for a blood vessel. Once they find one, the stylets penetrate the blood vessel, and the louse begins to suck blood. Their teeth serve as anchors in the skin. Three species of Anoplura occur in humans: Pediculus humanus and P. capatis, the human body and head louse, and Phthirus pubis, the

crab louse.

Slide: Phthirus pubis.

Slide: Pediculus humanus. Normal infestation with lice is little more than embarrassing but P. humanus can be the vector for typhus (disease agent: Rickettsia prowazecki), louse-borne relapsing fever (disease agent: Borellia recurentis), and trench-fever (disease agent: Bartonella quintanna).

Typhus Typhus is a rickettsial disease caused by Rickettsia prowazecki. Typhus has probably been more important in human history than any war. In World War I there were 3 million typhus-related deaths in Europe. Mortality from epidemic typhus ranges from 10-100% in any one outbreak. Humans are the only host of Rickettsia prowazecki, and the louse that vectors it, P. humanis, is generally only found on humans. The louse becomes infected when feeding on an

infected human. In the louse, the rickettsia will multiply to high numbers in the cells of the gut and is transmitted to humans either through the feces of the louse or when a louse is crushed on the skin. The disease agent enters the body either through a break in the skin or through the membranes in the eye, nose, or mouth. The rickettsia can remain alive in the louse for up to 60 days, so an area can remain contaminated for a long time. Thus we have a situation where a low-grade infection in a population erupts when conditions are favourable for it.

Class Arachnida: Subclass Acari – The mites and ticks Mites and ticks belong to the subclass Acari within the class Arachnida. Acari are characterized by an external reduction to two body segments: the capitulum (head), with its mouthparts; and the idiosoma with the internal organs and eight legs. Although ticks are essentially large mites, there are two main differences between ticks and mites; (1) ticks are much larger than mites and (2) they have an exposed toothed hypostome (the way in which they attach to their hosts).

Order Ixodida - Ticks: Ticks are important arthropods not only because they feed on the blood and tissues of humans and many other animals, but also because they transmit a large number of disease agents, including viruses, rickettsia, bacteria, apicomplexans, and filaroid nematodes. Ticks are pool feeders. This means that once attached to the host, the tick will inject saliva into the wound, which liquefies the tissues and allows them to suck up the fluid. Ticks sense CO2, warmth and movement from animals; when they sense an animal is nearby, they move toward the potential food source. Ticks are divided into two families: Ixodidae (hard ticks) and Argasidae (soft ticks). The head of a hard tick is visible when viewed from the dorsal side, where as the head of a soft tick is hidden beneath the body. In addition, hard ticks have a hardened dorsal sclerite or scutum which Argasids lack. You will only examine ticks in the family Ixodidae.

Family Ixodidae Adult ticks in this family range from 3-10 mm prior to blood feeding; the males do not increase much in size on feeding, but the females may be so engorged with blood, their abdomens become distended and their legs barely reach the ground. Development involves a series of feedings and moltings. The adults copulate while on the host, the females then drop off, lay eggs, and die.

Ixodid ticks are referred to as one-host, two-host, or three-host ticks. This refers to the number of animals on which a tick feeds during its life cycle. Knowledge of the number of hosts used by a tick is important for planning control programs and the understanding the epidemiology of the disease agent being transmitted by the tick.

Preserved sample: Boophilus annulatus: is an example of a one-host tick, whose principle host is cattle, though they have been found on deer and other large ruminants. B. annulatus is the vector of the apicomplexan parasite, Babesia bigemina, the cause of Texas red water fever. Infected ticks introduce sporozoites into the blood of its host. The sporozoite enters the erythrocytes, develops into trophozoites (merozoites) and multiplies by binary fission. Merozoites will differentiate into gamonts

within the red blood cells. Ticks become infected when they feed on the infectious

blood. Gamogany and fertilization occur in the gut of the tick. The kinete will ten migrate to the salivary glands where it can be passed on to the next vertebrate host.

Preserved sample: Ixodes scapularis, often referred to as the deer tick, is a three host tick that relies on rodents (Peromyscus spp.) as larvae and nymphs but moves to deer as adults. It is the vector of Lyme disease, caused by the spirochete Borrelia burgdorferi. This spirochete normally infects mice but can transfer to humans when humans replace deer as the final host of an infected tick.

Preserved sample: Dermacentor andersoni - the Rocky Mountain Wood tick is a three-host tick that uses mammals such as small rodents, lagomorphs, porcupines, large herbivores, canids and humans as hosts. It tends to feed on larger animals as it moves from larva to nymph to adult. The adults seek out hosts by questing; they climb on vegetation and hang head down with legs extended to catch on passing animals. D. andersoni is the vector for the causative agents of Colorado Tick Fever (viral disease that cycles between ticks and small rodents) and Rocky Mountain Spotted Fever (caused by Rickettsia rickettsi, cycles between ticks and small rodents).

Learning Objectives

1. Know general characteristics - Know differences between Insecta + Arachnida!!! - What character(s) of some arthropods allows them to be effective vectors?

2. Class Insecta

- General characteristics + anatomy 3. Oder Diptera

- Tsetse fly: Glossina - Visual id + know parasite and disease association

4. Family Culicidae - Mosquitoes

- Parasite and disease association - Life cycle - How can they find their hosts? - Behavioral characteristics that allow them to be effective vectors

5. Family Psychodidae – Sand flies

- Parasite and disease association - Visual id

6. Family Simulidae – Black flies

- Habitat - Visual id - Characteristics that make them good vectors - Parasite and disease association

7. Family Tabanidae – Horse flies

- Visual id - Parasite and disease association

8. Order Hemiptera

- Know general characteristics - mouthparts

9. Rhodnius prolixus

- Lifecycle - What habits allows them to be effective vectors? - Why might these habits affect transmission in lower income areas? - How do they find their host?

10. Family Cimicidae - Bed bugs

- Why might these insects be good vectors? - Are they vectors?

11. Order Siphonaptera – Fleas - Know they are parasites themselves - Life cycle, host, how do they find their host - What makes them good vectors? - Xenopsylla male and female: visual id - Parasite and disease association - Transmission - Plague cycle

12. Order Mallophaga – Lice

- Parasites themselves - How do the attach to host? - Life cycle - Chewing vs. sucking lice - Visual id Pedicularis humanus + Phthirus pubis - Parasite and disease associations - Typhus!

13. Class Arachnida

- Know general characteristics - Know general characteristics of ticks: families, feeding…

14. Order Ixodidae - Ticks

- Life cycle - 1,2,3 – host life cycles - Development - No Boophilus visual id but know parasite and disease associations etc.

15. Ixodes scapularis

- Visual id - 3 host life cycle - Parasite and disease association

16. Dermacentor andersoni

- 3 host tick - Disease and parasite association - Habits - Hosts