BIO 7505, Biology: A Molecular Approach, Laboratory
Mosquitoes, Dirofilaria immitis, Wolbacchia pipientis and WO Phage
Endosymbiotic But Deadly Relationships
One of the most interesting relationships in biology are endosymbiotic relationships, where organisms live in another
organism and evolve together. Where endosymbiosis and infectious diseases meet, the organisms can develop elegantly
woven lifecycles that impact the development of disease. One of these remarkable relationships is the relationship between
mosquitoes that carry heartworm. Mosquitoes transmit heartworm to dogs and cats and live off their hosts. Thus heartworm
is considered to be a parasite. The heartworm parasite (Dirofilaria immitis) is infected with the bacteria (Wolbacchia
pipientis) and the bacteria is infected with a virus (WO phage). The mosquito, heartworm, bacteria and virus have coevolved
together in remarkable ways that appears to mostly be beneficial to all of them. Thus they are considered to be in an
endosymbiotic relationship. In fact, they have evolved so tightly together that when dogs infected with W. pipientis (+)
heartworm are treated with antibiotics, the heartworm as well as the bacteria die! As will be discussed later, the dead
bacteria release substances that can lead to shock and death in their patient. It is very important for a vet to know if a
heartworm patient has heartworm infected with the bacteria!
To understand the relationships and organisms we are going to research, it is important to discuss all 3 organisms and the
WO phage independently from each other.
I. Mosquito Life Cycle and Ecology
A. Introduction to Mosquitoes
Mosquitoes are insects, and thus, have a multi-faceted life cycle intimately integrated into their ecosystems. To learn about
mosquito biology and ecology, go to the following link http://mosquito.ifas.ufl.edu/Mosquito_Life.htm, and read the three
sections on Mosquito Biology, Mosquito Ecology, and Mosquito Habitats. Even though this website is based out of Florida,
the information is still relevant to us in New York. After reading the three sections, write a brief summary of each of those
sections below. Remember, a summary is a concise reiteration of the most salient information.
Mosquito Biology:
1
Mosquito Ecology:
Mosquito Habitats:
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B. Mosquito Eradication
Because of their potential to spread disease and cause significant biological and economic harm, some scientists advocate for
complete eradication of mosquitoes! Other scientists believe such a drastic action could have serious consequences to our
planet’s biosphere. To decide what you think, go to the following link
http://www.nature.com/news/2010/100721/full/466432a.html and read: “A World Without Mosquitoes” (Published online
21 July 2010 | Nature 466, 432-434 (2010) | doi:10.1038/466432a). After you have read the article, write a brief summary
below of the arguments for and against mosquito eradication. Then, think critically about both sides of this argument, decide
which you think is most persuasive, write down your decision, and explain why you made that decision.
A World Without Mosquitoes?:
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II. Filariasis
Many animals are susceptible to filarial worm infection (known as filariasis), including humans, dogs, cats etc. They can cause
a wide variety of diseases, including heartworm, elephantiasis, lymphatic filariasis, etc. Human filariasis is a serious problem
in tropical region where people are exposed to mosquitoes for much of the year. Here in North America, our exposure is
limited by cold winters, which is thought to limit the proliferation of mosquitoes and thus the dissemination of these
diseases.
A
B
C
Figure 1- Human Filariasis: (Panel A)- The filariae that causes elephantiasis. (Panel B) A person with filarial infection
of the legs. (Panel C)- Human Filariasis Lifecycle
(www.cdc.gov)
(http://www.bing.com/images/search?q=filariasis&view=detail&id=A0CE66BE1775BE4CCC480044AE63446A96A1724E)
Figure 2- Joseph Merrick (The Elephant Man) Had Elephantiasis
(http://www.squidoo.com/elephantiasis)
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III. Dirofilaria

Go to www.ZipcodeZoo.com and type in “D. immitis” and “D. repens”.
You will see what is listed below. It is basically the taxonomic classification of the organism.
Kingdom: Animalia ( ) - animals
o Phylum: Nemata ( )
 Order: Spirurida ( )
 Family: Onchocercidae ( )
 Genus: Dirofilaria ( )
 Specific name: immitis - (Leidy, 1856)
 Scientific name: - Dirofilaria immitis (Leidy , 1856)

1)
2)
3)
Click on “Dirofilaria”
You will see that there are 3 species in this genus : D. immitis · D. repens · D. tenuis
Note that they belong to the phylum Nemata (nematodes or in lay terms round worms)
Click on “Nemata” the online list like the one above
a. Describe the habitats and basic anatomy of nematodes below
Nematode Habitat:
Nematode Anatomy:
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IV. Dirofilaria immitis Causes Canine and Feline Heartworm
 causes heartworm in dogs, cats and rarely people
 Our research will focus on this organism
Figure 3- Two Pictures of Heartworm in Dog Heart Post Mortum Examination
(http://www.bing.com/images/search?q=D.+immitis+and+picture&view=detail&id=117A64EA3261F3C0B999DFF406603A0FD3FDDE6E&first=1)
(http://images.google.com/imgres?imgurl=http://plpnemweb.ucdavis.edu/nemaplex/images/Dimmitis.jpg&imgrefurl=http://plpnemweb.ucdavis.edu/nema
plex/Taxadata/Dimmitis.htm&usg=__iNU0aLIKq7pL17EH_dvFG_wDR8M=&h=492&w=672&sz=46&hl=en&start=7&sig2=UzVvMov3EljpGNTqgHaIQ&zoom=1&tbnid=V166MHpjv 9wp0M:&tbnh=101&tbnw=138&ei=sQXqT-n4DOfE0AGYntXjDg&prev=/images%3Fq%3DDirofilaria%2B
immitis%26hl%3Den%26tbm%3Disch&itbs=1)

The heartworm lifecycle as presented by the Centers for Disease Control (CDC) is shown below in Figure 3
Figure 4- The Life Cycle of D. Immitis
(www.cdc.gov)
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
Go to the following website (http://www.metapathogen.com/heartworm)
1) Note that the organism does not just sit in the mosquito. It penetrates the midgut (insect stomach) and migrates to
the Malpighian tubules (insect kidney). During this process the nematodes progress through stages of its lifecycle
numbered L1 to L5 by molting.

Use the internet to define “molting” and write the definition below.
2) Note the changes in the nematode as they go through each stage of the lifecycle. L3 is the infectious stage that is
transmitted by mosquitoes. In this stage the nematodes migrate to the probiscus of the mosquito, and when it feeds
the worm is transmitted to the host. Once the worms reach the host (usually a dog or cat) they migrate and stick to
major blood vessels (especially the pulmonary artery), where they develop into the mature worm that causes
heartworm disease. When the heartworms grow they infiltrate the heart and cause the disease. They also release
more microfilaria into the blood stream of the host, which is taken-up by the next mosquito to continue the lifecycle.
V. Other species of Dirofilaria include D. repens and D. tenuis
D. repens
 usually only causes a skin infection, but is evolving and may be also involved in heartworm
D. tenuis
 causes disease in raccoons but is not thought to cause heartworm
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VI. D. immitis and Wolbacchia pipeintis
Figure 5- Insect Sperm Infected with W. Pipientis (green)
(http://eol.org/pages/976559/overview)
Wolbacchia pipientis is an intracellular bacteria that may infect as many as 20-60% of all insects worldwide. Their discovery in
the mosquito species Culex pipiens came with no fan fare or attention in the early 20th century. But now they are recognized
to play an important role in insect biology. Wolbacchia are currently organized into more than 4 supergroups (others call
these clades) according to their ability to infect certain types of organisms, which are designated A-D. Clades A and B infect
arthropods, while clades C and D infect nematodes, like D. immitis. And Wolbacchia has a real impact on insect evolution!
Go to www.ZipcodeZoo.com and search for Wolbacchia pipientis. Answer the following questions:
1) What role does this bacteria play in sexual differentiation? Be sure to look-up the definitions of cytoplasmic
incompatibility, speciation and parthenogenesis.
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2) How does W. pipientis confer evolutionary advantages?
VII. D. immitis and Wolbacchia pipeintis- The Clinical Significance
Additionally, W. pipientis has a real impact on heartworm disease. Dogs that are infected with W. pipientis (+) heartworms
are susceptible to toxic shock caused by the release of endotoxins into the bloodstream. Endotoxins are cell wall constituents
of so-called gram (-) bacteria, like lipopolysacchardies. Look at Figure 6 below and note the position of the capsule, cell wall
and plasma (cell) membrane in this general diagram of a prokaryote.
Figure 6- A General Diagram of a Prokaryote
(http://www.clker.com/cliparts/a/d/f/9/12554624301737124769Average_prokaryote_cell.svg.hi.png )
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1) What are Gram Negative bacteria? How do they differ from Gram Positive bacteria?
Toxic shock caused by endotoxins from W. pipientis happens two ways:
1) When the dog is treated for heartworm, a large number of worms die and release the endotoxin into their
bloodstream.
2) Alternatively, a vet may nick a worm during surgical excision of the worms and release a large amount of bacteria.
The net effect is a powerful activation of the dog’s immune response that causes the animal to go into shock. A vet really
should have prior knowledge of whether or not their patient is infected with heartworm carrying w. pipientis.
VIII. Wolbacchia pipientis and WO Phage
One of the most remarkable realizations of modern biology is that DNA really can change and that these changes drive
evolution. Changes in DNA are known as mutations and they may be bad or good for the survival of the organism. Regardless,
the accumulation of changes in DNA drives evolution.
There are many ways in which DNA can change. Single changes in the sequence of the DNA are known as “point mutations”,
which will be covered in more detail later in the semester. However, what many don’t realize is that entire blocks of DNA
move within a DNA molecule and even between chromosomes. Sometimes this causes sequences of DNA to be duplicated,
deleted or inverted (known as duplciation, deletion and inversion mutations, respectively). The primary mechanism for
these types of mutations is from viruses. In fact more than 50% of the human genome came from so called retroviruses. Our
ancestors were infected with these retroviruses and the viral DNA sequences were inserted into our own genomes and the
sequences were passed to the next generation.

Go online and look-up information about retroviruses.
1) Name a type of human disease caused by retroviruses.
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2) If a skin cell is infected with a retrovirus and the retrovirus inserts its DNA into the skin cell DNA, will it be passed
on to the next generation? Why or why not?
These moving sequences are known as transposons and the transposon DNA actually encodes an enzyme called a
transposase that allows the DNA to remove itself and insert itself somewhere else in chromosomes (i.e. to be mobile in your
DNA). These sequences have evolved further in us, and changed. Some transposons still encode a transposase and some no
longer have it.
Bacteria have viruses that infect them which are called bacteriophages or simply phages for short. Phages have a head which
contains its DNA, a sheath (Body) which is like a syringe and long legs which allows it to attach to bacteria (Figure VIII).
Figure VIII. Structure of a Bacteriophage
(http://www.bing.com/images/search?q=lifecycle+of+bacteriophage& view=detail&id=E5BA9166EAE2D5281E358196800970AFC42C2A5B)
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Bacteriophage inject their DNA into the bacteria once the virus attaches to the surface of the prokaryotic cell.
Figure IX. Picture of a Bacteriophage Injecting Its DNA Into a Bacterial Cell
(http://www.bing.com/images/search?q=Bacteriophage+Virus+Models&view=
detail&id=BF67E997FBFA537AAF044F6C39DB20F7DA7FE4E4&first=36)
Note orange bacteriophage, the green cell wall and the shortened sheath as the virus
prepares to inject its DNA (indicated by the black arrow).
Once the phage DNA is injected into the cell, one of two things can happen (Figure X):
1) The virus can chose the lytic lifecycle and create more of itself and break-open
(lyse) the cell.
2) Alternatively the viral DNA can be inserted into the circular bacterial genome and
thus chose the lysogenic lifecycle.
Figure IX- The Lytic and Lysogenic Lifecycle of Bacteriophage. Note that the bacterial chromosome is circular (purple) and in
the lysogenic lifecycle the red bacteriophage DNA (now called prophage DNA), is inserted into the bacterial chromosome.
(http://www.bing.com/images/search?q=bacteriophage+lytic+and+lysogenic+cycle&view=detail&id=CF07E9E1BB8C2AF3D20DA21A1387BE458EB2AAAF&first=32)
So while there are many differences between retroviruses and bacteriophages, bacteriophages act like retroviruses and insert
their DNA into bacterial chromosomes. The WO phage is no different from this. In fact 5 phage genomes can be identified in
some strains of the bacteria and it is estimated that upwards of 70% of the W. pipientis bacterial DNA is from a WO phage
(Klasson et al Mol Biol Evol. 2008 Sep;25(9):1877-87). Naturally, this means that bacteriophages have a huge impact on the
evolution of bacteria. How and why WO phage has impacted evolution of the organisms in this endosymbiotic relationship is
still being unraveled.
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