Vector Competence of Aedes sierrensis and Culex pipiens

University of the Pacific University of the Pacific

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University of the Pacific Theses and Dissertations Graduate School

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Vector Competence of Aedes sierrensis and Culex pipiens Vector Competence of Aedes sierrensis and Culex pipiens

complex (Diptera: Culicidae) for Dirofilaria immitis (Spirurida: complex (Diptera: Culicidae) for Dirofilaria immitis (Spirurida:

Onchocercidae) in Northern California Onchocercidae) in Northern California

Jeffrey Allan Kurosaka University of the Pacific

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Recommended Citation Recommended Citation Kurosaka, Jeffrey Allan. (2017). Vector Competence of Aedes sierrensis and Culex pipiens complex (Diptera: Culicidae) for Dirofilaria immitis (Spirurida: Onchocercidae) in Northern California. University of the Pacific, Thesis. https://scholarlycommons.pacific.edu/uop_etds/2979

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VECTOR COMPETENCE OF Aedes sierrensis AND Culex pipiens COMPLEX

(DIPTERA: CULICIDAE) FOR Dirofilaria immitis (SPIRURIDA:

ONCHOCERCIDAE) IN NORTHERN CALIFORNIA

by

Jeffrey Allan Kurosaka

A Thesis Submitted to the

Office of Research and Graduate Studies

In Partial Fulfillment of the

Requirements for the Degree of

MASTER OF SCIENCE

College of the Pacific

Biological Sciences

University of the Pacific

Stockton, California

2017

2




by


APPROVED BY:

Thesis Advisor: Tara Thiemann, Ph.D.

Committee Member: Shaoming Huang, Ph.D.

Committee Member: Kirkwood Land, Ph.D.

Department Chair: Craig Vierra, Ph.D.

Interim Dean of Graduate Studies: James A. Uchizono, Pharm.D., Ph.D

3




Copyright 2017

by


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DEDICATION

This thesis is dedicated to my friends and family, without whom none of this would have

been possible. To my best friend, Tommy Bolton, our friendship is something I could

not do without. You believed in me when even I had my doubts. Someday, I will find a

way to repay you for all that you have done for me. To my parents, Donald and Marilyn

Kurosaka, you were my teachers before I even knew the definition of the word, before I

even took my first breath. You taught me right from wrong and made me who I am

today. No matter what I do in life, it will never be enough to show my gratitude for all

that you both have sacrificed. As such, know that my accomplishments are your

accomplishments. Knowing that you are both proud of me is all the satisfaction that I

will ever need. To my older brother and sister, Douglas and Lisa Kurosaka, you have

protected and watched over me since I was born. I want you to know that I appreciate

you both not only for who you are, but for everything you have done for me whether I

knew about it or not. Life has thrown many curveballs my way. This document is a

testament to the difference all five of you have made in my life.

5

ACKNOWLEDGEMENTS

My appreciation goes to Dr. Tara Thiemann. Although I have had many teachers

throughout my academic career, she is by far the only one I consider my mentor. Over

the last two years, her support and guidance is what helped make this project a reality.

Additionally, I would like to thank Dr. Brittany Nelms for collaborating with us. Her

knowledge and assistance were invaluable throughout this project. I would also like to

express my gratitude toward the Lake County Vector Control District Board of Trustees

and the University of the Pacific’s Pacific Fund for funding my research. The Lake and

San Joaquin County Vector Control Districts for providing all the mosquitoes. The

Marin-Sonoma Mosquito and Vector Control District for allowing us to use their

facilities. Innovative research would not be possible without the support and

collaboration of the community. Special thanks to Dr. Shaoming Huang and Dr.

Kirkwood Land for agreeing to be on my thesis committee. Their input and feedback

were irreplaceable. It was a pleasure working alongside you both and perhaps in the

future I will have the honor of doing so again. Lastly, I would like to thank the

University of the Pacific, Department of Biological Sciences, for providing this amazing

opportunity and making all this possible.

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Vector Competence of Aedes sierrensis and Culex pipiens complex (Diptera: Culicidae)

for Dirofilaria immitis (Spirurida: Onchocercidae) in Northern California

Abstract

By Jeffrey A. Kurosaka

University of the Pacific

2017

Dirofilaria immitis Leidy (dog heartworm) is a life-threatening parasite transmitted

by mosquitoes to domestic dogs. Endemic in the eastern United States, cases have

become more prevalent over the last few decades. While prevalence in California is

generally low, Lake and San Joaquin Counties have reported rates comparable to the East

Coast at 3.73% and 0.71%(CAPC 2017), respectively. Aedes sierrensis is thought to be

responsible for transmission in California, but in some cases, it exists in inadequate

quantities and temporal ranges to explain parasite activity. Based on Huang et al. (2013)

and Tran (2016), bloodfeeding patterns, and other vector criteria, Culex pipiens complex

and Culiseta incidens were chosen to evaluate for vector competence. Female field-

caught mosquitoes were reared, infected (2.5-5 mff/μl), and decapitated at 15, 18, or 21

days post infection (dpi). Cs. incidens was reluctant to feed using an artificial feeding

system and will require additional trials. On the contrary, trials on Ae. sierrensis and Cx.

pipiens complex were both completed successfully. Both species were determined to be

competent vectors of D. immitis. Based on our findings, more than half of Ae. sierrensis

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females produced emerging L3s by 21 dpi, while Cx. pipiens complex never

produced L3s in more than 5% of females. In conjunction with other factors such as the

detection of D. immitis in wild mosquitoes, host-seeking preferences for domestic dogs,

and appropriate temporal overlap, this suggests that both Ae. sierrensis and Cx. pipiens

complex may play central roles in Lake or San Joaquin Counties, CA when abundant.

Targeted control efforts are necessary to reduce the incidence of canine heartworm in

these areas. While Lake and San Joaquin Counties, CA were the focus of this study, our

results may be applicable to the western United States when these species are relevant.

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TABLE OF CONTENTS

LIST OF TABLES………………………………………………………………………...9

LIST OF FIGURES…………………………………………………………………..….10

CHAPTER

1. Background Information

Canine Heartworm………………………………………….………..11

Life Cycle………………………………………………….…………12

Potential Hosts……………………………………………….………17

Therapy in Canines…………………………………………………..18

Vector Control………………………………………………….…....21

2. Vector Competence of Aedes sierrensis and Culex pipiens complex (Diptera:

Culicidae) for Dirofilaria immitis (Spirurida: Onchocercidae) in Northern

California

Introduction………………………………………………….……….22

Methodology…………………………………………………………25

Results………………………………………………………………..31

Discussion……………………………………………………………39

REFERENCES…………………………………………………………………………..49

9

LIST OF TABLES

Tables Page

2.1 Results of mosquitoes artificially infected with two titers of D. immitis (2016-

2017). Body parts (Abdomen/Head-Thorax) were tested for the presence of D.

immitis via PCR. Infective Rates refer specifically to the percentage of samples

that produced emerging infective larva(e) in warm PBS. Infected Rates refer to

the percentage of samples within each trail where D. immitis was detected in

either the abdomen, head-thorax, or warm PBS post-decapitation……..………..38

10

LIST OF FIGURES

Figure Page

1.1. Development of D. immitis within the mosquito. Arrows depict the pathway

followed by D. immitis as it progresses throughout the mosquito. S.G., Salivary

Glands. M.T., Malpighian Tubules.………………...............................................14

1.2. Complete life cycle of D. immitis, from microfilarial development in the

mosquito (mff-L3) to its infection of the host (Dog/Human/etc.).…...………….17

2.1. Geographic distribution of collection sites in Northern California between 2016-

2017. (A) Lake County, CA (B) San Joaquin County, CA………………............28

2.2. Third-stage larval (circled) emergence from a decapitated female mosquito

under a dissection microscope...……………………………………....................30

2.3. Seasonal dynamics of mosquito collection (left Y-axis) and 30-day HDU

accumulations (right Y-axis) between 2005-2015 in Lake County, California.

Threshold refers to the 130 HDUs required for D. immitis development. (A) CO2

traps (B) Resting collections………………………………………...…….……..34

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Chapter 1: Background Information

Canine Heartworm

Canine heartworm, otherwise known as D. immitis, is a parasitic nematode that

derives its name from the primary definitive host (domestic and wild canids) and organ

(heart) in which reproduction occurs(Cullens 2008). Belonging to the superfamily

Filarioidea, heartworms are often described as filarial, or thread-like, in nature and cause

a disease known as filariasis(Anderson 2000; Lok, Walker, Scoles 2000). Dirofilaria

immitis is one of many filarial nematodes known to affect public health. Others include

Wuchereria bancrofti Cobbold and Brugia Malayi Brug, Loa loa Cobbold, and

Mansonella Manson, which are responsible for lymphatic, subcutaneous, and serous

cavity filariases, respectively(CDC 2013; CDC 2015; CDC 2016).

Morphologically, heartworms are slender, white, and vary in size depending on

their sex and stage in development, with adults reaching lengths of approximately one

foot(Taylor 1960). Completion of a heartworm’s life cycle can take between 6 and 9

months in the canine host, with infections remaining patent up to 7.5 years(Abraham

1988; Anderson 2000; Newton 1968).

Heartworm disease, or dirofilariasis, is the manifestation of chronic symptoms

that result from the physiological burden that heartworms place cardiovascular system of

the definitive host. If left untreated, the infection can be fatal(Calvert, Rawlings, McCall

1999). Heartworm disease remains one of the most serious parasitic diseases

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affecting domestic dogs in North America and perhaps the world(Bowman and Atkins

2009; Simón et al. 2012).

Life Cycle

Heartworm transmission occurs when a susceptible female mosquito ingests an

infected bloodmeal containing microfilariae (early stage larvae, mff) from a definitive

host, supports development of the parasite to its infective stage, and transmits it to

another receptive, definitive host. Transmission requires a vector (carrier of transmission

for the parasite) to take at least two separate and appropriately timed bloodmeals. The

first must contain the microfilarial infection, while the second must be staggered

sufficiently to allow infective stage larval development(Cancrini and Gabrielli 2007). If

a female mosquito cannot accomplish this, then the parasite will not be

transmitted(Simón et al. 2012).

Development in the intermediate host. It is a common misconception that all

mosquitoes bite. In fact, both male and female mosquitoes can survive on water and

sucrose alone. Blood is required only by the female as a means of acquiring the protein

necessary to develop their eggs. Exceptions to this include autogenous species, which

utilize protein reserves accumulated during their larval stages to produce their first batch

of eggs. By comparison to anautogenous species that can require several blood meals for

egg-laying, autogenous species are considered relatively less important as vectors as their

chances of larval uptake and transmission is reduced(Cancrini and Kramer 2001;

Cancrini and Gabrielli 2007).

A mosquito species is considered a competent vector if it supports development of

the parasite to its infective stage. Development occurs in a series of stages. Each stage is

13

characterized by a molt and a migration, but not necessarily in that order(Kartman 1953a;

McCall et al. 2008). Transmission begins when a susceptible female mosquito ingests a

bloodmeal containing microfilariae. The microfilariae flow through the pharynx and into

the midgut along with the blood (Figure 1.1). Over the course of 24 hours, the

microfilariae escape the blood bolus and make their way into the Malpighian tubules

(M.T.) via its junction with the midgut. Upon reaching the distal end of the lumen, the

microfilariae invade the large, primary cells and transform into “sausage” stage larvae.

Although this is often considered the worm’s first larval stage (L1), this is a misnomer as

ecdysis (molting) does not occur at this time. By the seventh day, the L1 migrate back

into the lumen of the Malpighian tubules to continue developing. Larvae molt into

second-stage larvae (L2) around 10 dpi and then third-stage larvae (L3) by day 13. Upon

completion of development into L3s, the larvae will perforate the distal ends of the

Malpighian tubules and migrate toward the head via the hemocoel (body

cavity)(Abraham 1988; Bradley, Sauerman Jr., Nayar 1984; Bradley and Nayar 1987;

Cancrini and Kramer 2001; Kartman 1953a; Manfredi, DiCerbo, Genchi 2007; Serrão,

Labarthe, Lourenço-de-Oliveira 2001; Taylor 1960). Third-stage larvae are considered

“infective” once they have reached the cephalic spaces of the head, the salivary glands

(S.G.), or the proboscis(Manfredi, DiCerbo, Genchi 2007; McCall et al. 2008; Montarsi

et al. 2015; Taylor 1960). Canine heartworm exhibits positive thermotaxis, which

confers a drive to migrate toward higher temperature gradients(Stueben 1954). During

the female mosquito’s next bloodmeal, the L3 will be drawn to the warmth of the

definitive host and burrow out of the mouthparts of the mosquito. Emergence can occur

from various locations on the mosquito’s proboscis (needle-like mouthpart), from the

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folded labium (lower lip) to the tip which is called the labellum(Abraham 1988). Third-

stage larvae are carried along with the mosquito’s hemolymph and become deposited

onto the skin of the definitive host near the feeding wound. Once the mosquito has

finished ingesting its bloodmeal and removes its proboscis from the host, the larvae will

enter the host through the residual feeding wound(Cancrini and Kramer 2001; Grassi and

Noe 1900; McGreevy et al. 1974). Mosquitoes that prohibit development, whether it be

through mechanical defenses or innate immunity, are considered refractory(Michalski et

al. 2010). Globally, over 60 species of mosquitoes have been shown to be susceptible to

infection, 13 of which exist in the United States(Bowman and Atkins 2009; Lok 1988;

McCall et al. 2008; Otto and Jachowski Jr 1980). Research is necessary to determine

which of these mosquitoes can support development of heartworms to their infective

stage.

Figure 1.1. Development of D. immitis within the mosquito. Arrows depict the pathway

followed by D. immitis as it progresses throughout the mosquito. S.G., Salivary Glands.

M.T., Malpighian Tubules.

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Heartworm development in mosquitoes that can support D. immitis is

temperature-dependent and proportional to the degree of thermal exposure above the

14oC threshold(Knight and Lok 1998; Lok and Knight 1998; McCall et al. 2008; Stueben

1954). The rate of development described above was based on mosquitoes held at 26-

27oC and 80% relative humidity (RH), which require 10-17 days(McCall et al. 2008;

Taylor 1960). As climates continue to warm, the seasonal activity and geographic range

of the parasite is expected to continue to increase(Genchi et al. 2009; Ledesma and

Harrington 2011; Otranto, Capelli, Genchi 2009; Sacks, Chomel, Kasten 2004).

Approximately 130 heartworm development units (HDU) are required to allow the

parasite to reach its infective stage within the mosquito(Knight and Lok 1998).

Heartworm development units are defined as the accumulation of thermal units within a

range of 14oC to 30.5 oC, regardless of if they are consecutive(Christensen and Hollander

1978; Knight and Lok 1998; Lok and Knight 1998). Ambient temperatures below the

threshold will cause development to cease and may cause larvae to withdraw back into

the mosquito(Stueben 1954). The degree-day calculation below factors in the average

daily temperature above the developmental threshold for canine heartworm to

approximate the number of HDUs accumulated(Ledesma and Harrington 2015).

Accumulated HDUs = Σ Average Daily Temperature – (14 oC)

Development in the definitive host. Third-stage larvae utilize the residual feeding

wound site as a portal of entry into the definitive host(Grassi and Noe 1900; McGreevy et

al. 1974). By 3 dpi, the larvae molt into fourth stage larvae (L4) within the surrounding

subcutaneous tissue. Between 50 and 70 dpi, larvae molt into fifth-stage immature adults

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(L5) and enter the bloodstream through the surrounding blood vessels. Immature adults

can be found in the heart as early as day 70(Grieve, Lok, Glickman 1983). Complete

migration and maturation of all worms is observed by 120 dpi. Mature worms are not

only 10 times larger than their previous, immature state, but also they are capable of

producing microfilariae via sexual reproduction(Lichtenfels et al. 1985; Lichtenfels,

Pilitt, Wergin 1987; McCall et al. 2008). Microfilariae can detected within definitive

host’s blood between 6 to 9 months post infection, producing a patent infection capable

of infecting susceptible mosquitoes(Abraham 1988; Grieve, Lok, Glickman 1983; Knight

and Lok 1998; Kotani and Powers 1982; Kume and Itagaki 1955; McCall et al. 2008;

Orihel 1961). A vertebrate host that is incapable of supporting development or producing

a patent infection is considered a dead-end host. Dead-end hosts are not at risk of

becoming an infectious reservoir or developing most of the symptoms associated with the

parasite(Grieve, Lok, Glickman 1983; Ledesma and Harrington 2011).

Figure 1.2 is a simplified representation of D. immitis’s life cycle(CDC ). Canine

heartworm’s life cycle is obligate to both its intermediate host and its definitive host for

survival. While many courses of action are available to treat infected definitive hosts,

prevention is by far our best option(Nelson, McCall, Carithers 2014).

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Figure 1.2: Complete life cycle of D. immitis, from microfilarial development in the

mosquito (mff-L3) to its infection of the host (Dog/Human/etc.).

Potential Hosts

While domestic dogs are the primary reservoir of infection for canine heartworm,

several other species are also at risk. Other receptive hosts include, but are not limited to,

wild canids such as coyotes, felids, ferrets, raccoons, sea lions, penguins, and

humans(Bowman and Atkins 2009; Grieve, Lok, Glickman 1983; Ledesma and

Harrington 2011; McCall et al. 2008; Sacks, Chomel, Kasten 2004; Sano et al. 2005;

Simón et al. 2009; Theis 2005). Only some of the above hosts are capable of developing

patent, communicable infections(Bowman and Atkins 2009).

Hosts infected with canine heartworm can remain asymptomatic for months or

even years, with some never becoming symptomatic at all. Factors that may influence

the development of symptoms include the species of definitive host, the density of filarial

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burden, individual reactivity, and the level of exercise(McCall et al. 2008). In dogs,

symptoms range from persistent coughing to lethargy, anorexia, and even

mortality(Calvert, Rawlings, McCall 1999; Simón et al. 2009; Simón et al. 2012).

Human cases, however, are less severe as they are incapable of supporting adult

heartworm development. Symptoms include of coughing, hemoptysis, chest pain, fever,

and sometimes pleural diffusion(Global Health - Division of Parasitic Diseases 2012;

Roy, Chirurgi, Theis 1993; Simón et al. 2009; Simón et al. 2012; Theis 2005).

Although human cases are not fatal, they often present diagnostic complications

for physicians. For example, heartworms are recognized on chest radiograms and CT

scans as coin lesions, or dense, circular masses with smooth edges(McCall et al. 2008).

While the diagnostic differential for a coin lesion varies, a common preliminary

interpretation is cancer(Allison et al. 2004; Theis 2005; Toomes et al. 1983; Trunk,

Gracey, Byrd 1974). Extensive clinical tests are necessary to determine the etiology of

the condition, the cost of which can exceed $80,000 per patient(Theis 2005). Meanwhile,

the patient is left to deal the possibility that they may have cancer when in fact the actual

cause of their coin lesion was a small pulmonary infarction (tissue death) caused by

heartworm-related embolic clots(Gómez-Merino et al. 2002; Rena, Leutner, Casadio

2002; Roy, Chirurgi, Theis 1993; Simón et al. 2012). Although none of the differentials

are desirable, treatment for the latter is preferred as it usually only requires a simple

procedure to surgical remove any nodules or worms present(Simón et al. 2012).

Therapy in Canines

Over the span of approximately 6 to 9 months, large quantities of adult

heartworms can obstruct blood flow, form clots, and produce infectious microfilariae

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within the vasculature of the definitive host(Anderson 2000; Rawlings et al. 1993).

Therapy can be very effective when it comes to preventing infection, averting further

damage, and precluding the development of additional symptoms. When it comes to

therapy, two types exist: preventative therapy and adulticidal therapy(Bowman and

Atkins 2009).

Preventative therapy. Preventatives are the best option for uninfected

dogs(Nelson, McCall, Carithers 2014). Providing protection with minimal side effects,

preventatives can be used in dogs as young as 8 weeks old(Cruthers et al. 2008; McCall

et al. 2001). Regimens range from monthly to yearly depending on the formula and

dosage. Avermectins and milbemycins are two series of commercially available drugs

that rely on a group complex parasiticidal compounds known as macrocyclic

lactones(McCall et al. 2008). Macrocyclic lactones kill larvae up to 60 days old(McCall

et al. 2001). A single dose has been observed to be 80% to 90% effective (Blagburn,

Paul, Newton 2001; Dzimianski et al. 2001; Lok, Knight, Ramadan 1989).

Currently, no other active ingredient other than macrocyclic lactones have been

approved as a prophylactic in dogs by the FDA. Caution and strict adherence to proper

usage is strongly advised to minimize the risk of resistance(Bowman and Atkins 2009;

Prichard 2005). While they have been used experimentally to prevent the development of

additional adult heartworms in already infected hosts, this is not recommended as

preventatives are no longer a viable option once the larvae have developed into

adults(Blair, Williams, Ewanciw 1982; Bowman et al. 1992; Lok, Knight, Ramadan

1989). As a result, macrocyclic lactones are not approved for safe usage in dogs with

adult heartworms or in dogs with significant symptoms(Lok, Knight, Ramadan 1989).

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Adulticidal therapy. Melarsomine dihydrochloride is the only treatment approved

by the FDA to treat adult heartworms(Bowman and Atkins 2009; Nelson, McCall,

Carithers 2014). Melarsomine dihydrochloride is an organoarsenic compound that acts as

an immiticide to kill adult heartworms and prevent further damage to the pulmonary

vasculature. Tests have shown that two doses given intramuscularly every 24 hours have

an efficacy of at least 96%. Repeated 4 months later, 99% of adult heartworms were

killed(Miller et al. 1995). While extremely effective, this method possesses several

shortcomings, including, but not limited to, the cost, the ill effects of arsenic exposure,

and the threat posed by dead worms. Dead heartworms can lead to inflammation,

thromboembolisms, arterial obstruction, and vasoconstriction(Kramer 2006).

Furthermore, melarsomine must be used in conjunction with tranquilizers over the entire

course of the regimen to reduce the circulation of the infection. While this does not

typically confer added risk, it does add to the expenses of treatment and require

continuous maintenance to keep the patient sedated(Miller et al. 1995; Nelson, McCall,

Carithers 2014).

Due to the risks involved with chemical methods, invasive surgery is not

uncommon. Extracting heartworms from anesthetized dogs using forceps has shown to

be 90% effective initially, with some patients dying from heart and renal failure post-

treatment. Unfortunately, follow-up treatments still require melarsomines to completely

cure the infection(Bowman and Atkins 2009; Morini et al. 1998; Nelson, McCall,

Carithers 2014; Sasaki Y, Kitagawa H, Ishihara K. 1989).

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Vector Control

Vector control is one option that shows promise to not only reduce the incidence of

heartworm disease, but also to limit the spread of all mosquito-transmitted infections.

Mosquito species important to transmission can be identified based upon their completion

of key vector identification criteria(Ledesma and Harrington 2011). Ranked in order of

importance, first the infection must be detected in wild-specimens. Second, the

geographic distribution of the mosquito must overlap sufficiently to explain the

prevalence of the infection in either wild of domestic animals. Third, the species of

mosquito in question must feed on relevant wild or domestic animals in nature. Fourth,

the mosquitoes must feed frequently on those same hosts in nature. Lastly, field strains

of the mosquitoes must demonstrate competence, or the ability to develop the infection to

its infective stage. This final criterion is typically evaluated in a laboratory, where

infection variables are easier to control. By completing these criteria, it is possible to

identify and target vectors of importance to transmission. Targeting vectors of

transmission interferes with the intermediate host’s ability to continue the life cycle of the

causative agent. This is especially useful when it comes to canine heartworm since all

potential reservoirs for canine heartworm have yet to be determined.

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Chapter 2: Vector Competence of Aedes sierrensis and Culex pipiens complex

(Diptera: Culicidae) for Dirofilaria immitis (Spirurida: Onchocercidae) in

Northern California

Introduction

Canine heartworm, caused by the filarial parasite Dirofilaria immitis, is almost

entirely preventable with oral prophylactics(Blagburn, Paul, Newton 2001; Dzimianski et

al. 2001; Lok, Knight, Ramadan 1989). Despite this, contracting the infection is

practically inevitable over the lifetime of an unprotected domestic dog in areas endemic

with the parasite(Nelson, McCall, Carithers 2014). According to the Companion Animal

Parasite Council (2017), 1.28% of dogs in the United States tested positive for D. immitis

in 2016. Unfortunately, this value does not represent the overall prevalence of the

parasite amongst domestic dogs as it pertains only to those tested for the parasite, 1

million of which came back positive((APPA) American Pet Products Association ; CAPC

2017). Estimates predict the actual activity of the parasite to be at least three times that

as most infections go undiagnosed(Genchi, Kramer, Rivasi 2011; IDEXX Laboratories

and ANTECH Diagnostics ). Furthermore, domestic dogs are not the only definitive

hosts susceptible to being infected. Heartworms have been recovered from coyotes and

other wild canids, felids, mustelids, ungulates, marine mammals, and humans

23

(Grieve, Lok, Glickman 1983; Ledesma and Harrington 2011; McCall et al. 2008; Sacks,

Chomel, Kasten 2004; Sano et al. 2005; Simón et al. 2009; Theis 2005). As a result,

prevalence of D. immitis worldwide is expected to be even greater than previously

estimated(IDEXX Laboratories and ANTECH Diagnostics ; McCall et al. 2008).

Failure to address the rising incidence of D. immitis has considerable

repercussions on public health, veterinary and human alike. Chronic infections can lead

to irreparable damage to the heart, lungs, and arteries in domestic dogs, which result in

persistent coughing, lethargy, anorexia, and in some cases, death(Calvert, Rawlings,

McCall 1999; McCall et al. 2008; Simón et al. 2009; Simón et al. 2012). In addition to

these effects in dogs, previous studies suggest that mosquitoes coinfected with other

filarial nematodes like D. immitis could transmit arboviruses, such as chikungunya virus,

at higher rates to humans and other animals(Vaughan and Turell 1996; Zytoon, El-

Belbasi, Matsumura 1993; Zytoon, El‐Belbasi, Matsumura 1993).

To mitigate these downstream effects and reduce the overall activity of D.

immitis, preventative and immiticidal therapy are both effective options(Bowman and

Atkins 2009; Cruthers et al. 2008; Hampshire 2005; Sasaki Y, Kitagawa H, Ishihara K.

1989; Vezzoni, Genchi, Raynaud 1992). Access to these options, however, is not always

possible. Mosquito vector control is an effective alternative capable of avoiding many of

the complications associated with D. immitis therapy or the lack thereof(Vezzoni, Genchi,

Raynaud 1992). By interfering in the parasite’s development within the mosquito, it is

possible to reduce the incidence of cases among all susceptible definitive hosts, not just

domestic dogs. While research has been completed in the United States, much of it is not

directly applicable as it pertains to the East Coast(Butts 1979; Lewandowski Jr, Hooper,

24

Newson 1980; Licitra et al. 2010; Lindsey 1961; Lowrie 1991; Parker 1986; Sauerman Jr.

and Nayar 1983; Thrasher, Ash, Little 1963; Thrasher 1968; Thrasher et al. 1968; Tolbert

and Johnson Jr. 1982; Villavaso and Steelman 1970; Wallenstein and Tibola 1960; Watts

et al. 2001). Mosquito biology varies from species to species as well as between different

localities(Knight and Lok 1998; Ledesma and Harrington 2011; Nayar, Knight, Bradley

1988; Tiawsirisup and Kaewthamasorn 2007). Research specific to the West Coast is

necessary to identify mosquito species important for parasite transmission in Northern

California.

Lake County and San Joaquin County were chosen as the sites of this study due to

their higher than average levels of parasite activity at 3.73% and 0.71%, respectively, in

2016(CAPC 2017). Previously, Aedes, Culex, and Culiseta mosquitoes were tested for

the presence of D. immitis in San Joaquin County, CA(Huang et al. 2013). Seven of the

fifteen mosquito species tested positive for this parasite. Culex pipiens complex and

Culiseta incidens were among them and have been chosen for further investigation based

on availability and their ability to fulfill key vector criteria(Ledesma and Harrington

2011). Culex pipiens complex (total number of specimen, n=40; minimum infection rate

estimates infection rates per 1,000 mosquitoes, MIR=3.66) was selected because it tested

positive for D. immitis in San Joaquin County, displays significant ecological overlap

with the parasite, possesses appropriate seasonality, habitually feeds on dogs, and

possesses a geographical distribution that could help explain activity in San Joaquin

County(Huang et al. 2013; Thiemann et al. 2012). Similarly, Culiseta incidens (n=11;

MIR=2.81) tested positive in San Joaquin County, exists in urban/residential settings, and

exhibits a strong tendency to feed on domestic dogs, which is imperative for parasite

25

transmission(Huang et al. 2013; Theis et al. 2000; Thiemann ). Aedes sierrensis (n=1;

MIR=6.7) was chosen as a positive control for this study since it has been shown to

support development in previous studies(Theis et al. 2000; Tran, Nelms, Thiemann 2016;

Walters and Lavoipierre 1982; Walters 1995). While Ae. sierrensis has been considered

the primary vector of D. immitis in northern California since 1974, ecological data

suggests that other mosquito species may be contributing to the observed activity due to

the limited potential of Ae. sierrensis in some areas(Huang et al. 2013; Weinmann and

García 1974).

The current study explored the potential of Ae. sierrensis, Cx. pipiens complex,

and Cs. incidens as vectors for D. immitis in Northern California by examining D. immitis

prevalence, mosquito abundance, and temperature trends(CAPC 2017; Lake County

Mosquito and Vector Control District 2005-2015; NOAA 2017). These mosquito species

of suspected importance were then evaluated based on their ability to support D. immitis

development to its infective L3 stage. Identifying vectors of potential importance to D.

immitis transmission should help vector control districts better target competent mosquito

species, reducing the overall incidence of canine heartworm disease.

Methodology

Mosquito abundance. Species-specific mosquito abundance data were obtained

for Lake County, CA from the Lake County Vector Control District and can be accessed

using CalSurv Gateway, a California Vector-borne Disease Surveillance System that

stores reported surveillance data from the California Department of Public Health, the

Mosquito and Vector Control Association of California, and the University of

California(CalSurv Gateway, UC Davis Center for Vector-borne Diseases 2017; Lake

26

County Mosquito and Vector Control District 2005-2015). Abundance data for each

species was compiled into 11-year (2005-2015) average daily collections per trap per

night to approximate species seasonality and relative abundance.

Mosquitoes were collected using the following methods: carbon dioxide (CO2)

traps, New Jersey light traps (NJLT), and resting collections. CO2 traps, which rely on

carbon dioxide to attract host-seeking mosquitoes, were only deployed between February

and October by comparison to the other methods which were conducted year-round.

New Jersey light traps use light to attract insects, including mosquitoes(Mulhern 1942).

Resting collections were actively collected using aspirators and then placed into

collection containers.

Temperature data. U.S. Climate Normals, which are three-decade averages

(1981-2010), were obtained from the National Oceanic and Atmospheric Administration

(NOAA) to estimate the average daily temperature in Lake County, CA throughout the

year(NOAA 2017). Temperature readings originated from a single station southeast of

Clearlake, CA. Although temperatures may vary throughout the county, Climate

Normals were chosen due to inconsistencies in data collection at other stations over time.

Heartworm development units (HDUs) were determined by calculating the 30 day

(average life-span of a mosquito) differential between the average daily temperature and

the development threshold for the D. immitis (14 oC)(Christensen and Hollander 1978;

Huang et al. 2013; Knight and Lok 1998; Ledesma and Harrington 2015; Lok and Knight

1998).

Accumulated HDUs = Σ Average Daily Temperature – (14 oC)

27

Mosquito collection/rearing. Mosquitoes were collected as egg rafts or larvae

from Lake (Ae. sierrensis and Cs. incidens) and San Joaquin Counties (Cx. pipiens

complex), California (Figure 2.1). Larvae were reared and maintained on tropical fish

food until emergence in an insectary. Adults were maintained on 10% sucrose solution at

26°C with a 14:10 (L:D) photoperiod.

Microfilaremic blood handling. Microfilaremic blood was obtained from the

National Institute of Health/National Institute of Allergy and Infectious Diseases

(NIH/NIAID) Filariasis Research Reagent Resource (FR3) Center (Missouri strain – Ae.

sierrensis, Cx. pipiens complex, & Cs. incidens) and from TRS Lab Inc. (Wildcat strain –

Ae. sierrensis). The Missouri isolate originated from an animal pound in Missouri in

2000, while the Wildcat isolate originated from Stanwood, MI and has been maintained

since August 2012.

To determine the initial titer of microfilariae present, the blood was first diluted

(1:10) and observed in 20µl dual-chambered plastic cellometer slides (Nexcelom

Bioscience, Lawrence, MA). Depending on the initial titer, the infected blood was either

diluted with rabbit blood or concentrated via centrifugation. Centrifugation of the blood

occurred in 15 ml centrifuge tubes at 4°C for 10 minutes at 1000g with a gradual

acceleration and deceleration, consistent with FR3 guidelines. The above steps were

repeated as necessary to achieve the desired 2.5 mff/μl low titer and 5 mff/μl high titer.

Microfilarial infections range within domestic dogs from 0.1 mff/μl to 50 mff/μl, with

most infections being around 10 mff/μl. The microfilarial range chosen for this

experiment was based on research by Lai, whom concluded that lower microfilarial

28

Figure 2.1A

Figure 2.1B

Figure 2.1. Geographic distribution of collection sites in Northern California between

2016-2017. (A) Lake County, CA (B) San Joaquin County, CA

29

densities may play a major role in transmission between dogs and mosquito vectors(Lai

et al. 2000).

Blood was then loaded (1-2 ml) into the reservoir of the Hemotek® feeder

(Discovery Workshops, Accrington, UK), which was covered with pig intestine and

sealed with a rubber o-ring. Assembled reservoirs were loaded onto to the heating unit

immediately prior to the infection.

Dog heartworm infection. Adult mosquitoes were transported to the Marin-

Sonoma Mosquito & Vector Control District (BSL-2 facility) for infection.

Approximately 1000 3-4 day old females were used in each infection. Mosquitoes were

starved of both water and sucrose solution 24-36 hours prior to bloodfeeding. Using a

Hemotek® artificial feeding system, starved mosquitoes were fed blood containing either

a low (1-3.5 mff/μl) or high titer (~5 mff/μl) of D. immitis microfilariae. After 1 hour,

engorged females were anesthetized (CO2 and ice) and separated into smaller, disposable

chambers to be maintained on water and rehydrated craisins in an incubator at 24.5°C and

86% RH. Immediately following the infection, midgut smears were analyzed to confirm

the presence of live microfilariae. This was completed on both the experimental species

and Ae. sierrensis, which was tested prior and acted as a control to confirm the success of

the infection.

Decapitation. At 15, 18 and 21 days post-infection, females were decapitated

under a trinocular dissecting microscope using double depression slides filled with warm

phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, and

1.47 mM KH2PO4) and an insect pin (Figure 2.2). Decapitated samples were incubated

at 37°C for 30 minutes to allow time for L3 emergence. The number of L3 larva was

30

recorded and whole mosquitoes (head and body) were transferred into individual 2.0 ml

safe-lock tubes. Samples were transported in coolers containing ice packs to the

University of the Pacific and stored at -80°C for further testing.

Figure 2.2. Third-stage larval (circled) emergence from a decapitated female mosquito

under a dissection microscope.

Molecular testing. Mosquito samples were dissected on glass microscope slides

using razor blades to separate the abdomen from the thorax. Afterwards, the thorax was

placed in the sample’s original tube along with the head, while the abdomen was placed

in a new safe-lock tube. DNA was extracted from the separated mosquito head-thoraces

and abdomens using DNeasy 96 Blood & Tissue Kit (Qiagen, Valencia, CA). Dirofilaria

immitis-specific 5s-sp primers (U.S. Patent No.: 6,268,153 Bl, forward sequence: 5’-

31

CAAGCCATTTTTCGATG CACT-3’, reverse sequence: 5’-CCATTGTACCGCTTAC

TACTC-3’) were used to detect D. immitis DNA (193-bp)(Huang et al. 2013; Lizotte-

Waniewski, Michelle (Northampton, Steven A. (North Hatfield, MA) 2001). Dirofilaria

immitis DNA extracted from infected blood (positive) and nuclease-free H2O (negative)

were tested alongside each set of experimental samples as controls. PCR amplification

was carried out in a 25 µl reaction mix containing 10x PCR buffer II, 2.5 mM of MgCl2,

0.2 mM dNTP, 20 mg/ml of BSA, 0.2 µM of each primer, 1 U of Hotstart AmpliTaq

Gold polymerase, 6 µl of DNA, and an appropriate volume of nuclease-free H2O.

Similar to Huang, PCR reactions cycled as follows: 10 min of 95oC; 35 cycles of 95 oC

for 15s, 52 oC for 30s, and 72 oC for 30s; and 10 min of extension at 72 oC. PCR products

were run on 1.2% agarose gels stained with GelRed dye (Biotium, Fremont, CA)(Huang

et al. 2013).

Sample size and statistics. Approximately 1000 mosquitoes were initially

included in each trial. This was done under the assumption that as many as half of these

mosquitoes would not feed and another half would not survive to the desired time points

(15, 18, & 21 dpi). Roughly 10-20 mosquitoes were decapitated at 15 dpi, 25-50 at 18

dpi, and the rest were decapitated at 21 dpi. Results were analyzed using chi-square, as

done in previous vector competence studies(Reisen, Fang, Martinez 2005).

Results

Relative mosquito abundance and HDU accumulation. From 2005-2015, a

total of 29,382 female Ae. sierrensis, Cx. pipiens complex and Cs. incidens were

collected using CO2 traps, New Jersey light traps (NJLT), and resting collections in Lake

County, CA(CalSurv Gateway, UC Davis Center for Vector-borne Diseases 2017; Lake

32

County Mosquito and Vector Control District 2005-2015). Combined, CO2 traps and

resting collections accounted for 99.1% of collections (68% and 31%, respectively). To

alleviate collection method bias, both CO2 traps and resting collections were considered

separately to determine the relative abundance and seasonality for each species of

interest. The number of mosquitoes captured from NJLT (0.87%) were negligible and

therefore excluded.

Of the three species under investigation, Ae. sierrensis were the most abundant

(n=24,338). CO2 traps accounted for 74.9% of collections for this species. The earliest

detection of Ae. sierrensis was in early-March. Abundance rapidly increased in mid-

April, peaked in a mid-May, and then gradually declined until the end of October. In

total, the majority (~98.7%) of female Ae. sierrensis were trapped between April and

September. This temporal trend was relatively consistent between CO2 trap collections

and resting collections.

Culex pipiens complex were the least abundant (n=391). When deployed

concurrently, both CO2 traps and resting collections were equally effective averaging

0.47 and 0.52 mosquitoes per trap per night between May and September. Culex pipiens

complex were collected inconsistently year-round. Abundance steadily increased in May,

peaked in August and November, and declined in December. Most females (~97.7%)

were captured between May and December.

Finally, 4,641 female Cs. incidens were collected. Again, both CO2 traps and

resting collections had comparable efficacies, catching 2.2 and 2.9 mosquitoes per trap

per night between May and September, respectively. Similar to San Joaquin County, Cs.

33

incidens were collected consistently year-round, peaking in July(Huang et al. 2013).

Most females (~91.9%) were collected between March and November.

The relative abundance and degree of seasonal overlap with HDU accumulation is

shown in Figure 2.3 using CO2 traps and resting collections from Lake County, CA. In

Lake County, CA, 130 or greater HDUs falls between late-June and mid-October (Week

26- 41). Ranked in order based on relative abundance, Ae. sierrensis, Cs. incidens, and

Cx. pipiens complex are each present during this period. As such, none of these three

potential vectors can be ruled out based on temporal overlap alone.

34

Figure 2.3A:

Figure 2.3B:

Figure 2.3. Seasonal dynamics of mosquito collection (left Y-axis) and 30-day HDU

accumulations (right Y-axis) between 2005-2015 in Lake County, California. Threshold

refers to the 130 HDUs required for D. immitis development. (A) CO2 traps (B) Resting

collections.

35

Molecular testing of abdomens and head/thoraces. Heads and thoraces were

tested together and abdomens were tested separately to determine the extent to which the

infection progressed within each species.

Positive bands for D. immitis DNA were easily detected in Ae. sierrensis.

Throughout the various trials, 40-67.85% of abdomens and 42.2-84.2% of head-thoraces

tested positive for D. immitis DNA. Overall, D. immitis DNA could be detected in 71%

or more of Ae. sierrensis that ingested the infected bloodmeal.

No detectable signs of D. immitis DNA were observed at any time point

regardless of the body part tested for Cs. incidens. Either the microfilariae were never

able to become established or any remnants of the parasite were digested and excreted.

Unlike Ae. sierrensis, bands for D. immitis DNA were difficult to detect in

samples extracted from Cx. pipiens complex as bands were particularly faint. Between

the two titers of the Missouri strain, up to 43.18% of abdomens and 63.5% of head-

thoraces tested positive for D. immitis DNA. The high titer resulted in significantly fewer

(χ2 = 20.77, df = 1, P < 0.001) PCR positives by the end of each experiment. Overall, D.

immitis DNA was detected in 77.1% of low titer mosquitoes and 32.6% of high titer

mosquitoes.

Vector competence. In total, 1,523 females representing three species, each from

different genera (Aedes, Culex, and Culiseta), fed on infected blood during this study

(Table 1).

Aedes sierrensis was infected with both the Missouri and the Wildcat strain of D.

immitis. Mosquitoes infected with the low titer of the Missouri strain initially displayed

no signs of infective stage larvae at 15 dpi. Over the course of the experiment this was

36

no longer the case as the infective rate increased significantly (χ2 = 11.04, df = 1, P <

0.001) from 0% to 55% by 21 dpi. Of the 38 infective females that survived to be

decapitated, an average of 2.7 L3s (range 1-7) emerged. To complement the previous

trial, both a low and a high titer of a comparable strain (Wildcat) were completed since

the FR3 facility was no longer able to supply adequate titers of the infected blood.

Mosquitoes infected with the low titer of the Wildcat strain resulted in more than half of

the mosquitoes being infective at any given time point. Of the 43 infective females

decapitated, an average of 6.1 L3 (range 1-23) emerged. By comparison, mosquitoes

infected with the high titer of the Wildcat strain demonstrated complete infectivity by 18

dpi. A mean of 10.5 L3 (range 2-35) emerged from the 46 decapitated infective

mosquitoes. Comparing the two strains at low titer, there was no significant difference in

infectivity at 21 dpi. Despite this, the survivorship of mosquitoes infected with the

Wildcat strain was significantly lower (χ2 = 16.25, df = 1, P < 0.001). Between the two

titers of mosquitoes infected with the Wildcat strain, there was a significant difference (χ2

= 7.42, df = 1, P = 0.006) in the infective rate of mosquitoes at 21 dpi. The increased

microfilarial density likely explains the significantly lower (χ2 = 6.85, df = 1, P = 0.008)

survivorship of the high titer trial. Regardless, all three infections of Ae. sierrensis

produced infective stage larvae.

Unfortunately, Cs. incidens were reluctant to feed using the Hemotek® artificial

feeding system. Although 22 females became partially or completely engorged and 11

were eventually decapitated, no L3s emerged. Simultaneously infected Ae. sierrensis

confirmed microfilarial uptake and provided some confidence in the results of this trial.

37

This was the only instance where the desired titer was not successfully achieved as the

blood used in this trail contained only 1.16 mff/μl.

Both a low and a high titer trial for the Missouri strain were completed for Cx.

pipiens complex. Mosquitoes infected with either titer displayed little to no signs of

infective stage larvae. Neither time nor varying titers resulted in a significant difference

(χ2 < 2.8, df = 1, P > 0.09) in observed infective rates or survivorship. Never more than 2

L3s were observed from infective mosquitoes. A mean 1.2 L3 (range 1-2) and 1 L3

emerged from the low and high titer, respectively. Aedes sierrensis confirmed

microfilarial uptake for the high titer trial.

38

Table 2.1. Results of mosquitoes artificially infected with two titers of D. immitis (2016-2017). Body parts (Abdomen/Head-Thorax)

were tested for the presence of D. immitis via PCR. Infective Rates refer specifically to the percentage of samples that produced

emerging infective larva(e) in warm PBS. Infected Rates refer to the percentage of samples within each trail where D. immitis was

detected in either the abdomen, head-thorax, or warm PBS post-decapitation.

D.i., Dirofilaria immitis. S.D., Standard Deviation of L3 larva(e) present.

* Percentages with different lowercase letter(s) indicate that they are statistically different by chi-squared test (P ≤ 0.05).

Strain

(Source) Species Titer

No.

engorged

Survivorship

(%) Dpi

Mosquitoes

tested

Abdomens

Positive

for D.i. (%)

Head-

Thoraces

Positive for

D.i. (%)

Infected

Rate (%)

Infective

Rate (%)*

# of

Infective

Mosquitoes

Mean Infective

L3s ± S.D.

15 11 45.4 45.4 72.7 0a 0 0

18 38 44.7 44.7 71 34.2b 13 2.85 ± 1.86

21 45 40 42.2 80 55b,c 25 2.68 ± 2.05

15 20 30 20 35 5a 1 1

18 44 43.18 31.8 59.1 4.5a 2 1.5 ± 0.71

21 96 41.7 63.5 77.1 1.1a 2 1

15 10 0 0 0 0a 0 0

18 26 3.2 6.4 12.9 3.2a 1 1

21 46 6.5 26.1 32.6 2.2a 1 1

Culiseta incidens Low 22 50 21 11 0 0 0 0a 0 0

15 15 66.7 66.7 80 53b,c 8 5.87 ± 7.64

18 26 50 53.8 100 73c,d 19 5.16 ± 4.27

21 21 52.4 57 95 76c,d 16 7.25 ± 6.42

15 19 63.1 84.2 94.7 94.7d,e 18 7.33 ± 3.53

18 28 67.85 57.1 100 100e 28 12.5 ± 9.31

56

64.2

72.5

34.4

22.5

Missouri

(FR3)

Low

Culex pipiens

complex High

High

Aedes sierrensis

Aedes sierrensisWildcat

(TRS) 209

Aedes sierrensis Low 168

Culex pipiens

complex Low 380

120

180

39

Discussion

Although competence in Ae. sierrensis has been demonstrated perviously(Walters

and Lavoipierre 1982), Huang et al. (2013) suggested that in regions like San Joaquin

County, CA where abundance and seasonal activity may be limited, other mosquito

species may be responsible for the observed prevalence in heartworm activity. This study

investigated the ability of various mosquito species to develop D. immitis in Northern

California to identify key vectors of transmission. Temporally, Ae. sierrensis, Cs.

incidens, and Cx. pipiens complex are present to varying degrees during the period in

which HDUs exceed 130. In Lake and San Joaquin Counties, CA, this period

corresponds with mid-June to mid-October and mid-March to November,

respectively(Huang et al. 2013). Combining this evidence with information pertaining to

other key vector identification criteria, such as the detection of D. immitis in wild

mosquitoes and host-seeking preferences associated with domestic dogs, the potential of

these three mosquito species as vectors of D. immitis in northern California cannot be

ruled out without information regarding their competence in the laboratory(Huang et al.

2013; Theis et al. 2000; Thiemann et al. 2012; Tran, Nelms, Thiemann 2016; Walters and

Lavoipierre 1982; Walters 1995; Weinmann and García 1974).

Dirofilarial infections begin in the midgut (abdomen), migrate to the Malpighian

tubules (abdomen), and become infective within the anterior (head-thorax)(Abraham

1988; Bradley and Nayar 1987; Cancrini and Kramer 2001; Kartman 1953a; Manfredi,

DiCerbo, Genchi 2007; McCall et al. 2008; Montarsi et al. 2015; Serrão, Labarthe,

Lourenço-de-Oliveira 2001; Taylor 1960). Meanwhile, the mosquito’s biology can

digest, degrade, or excrete traces of D. immitis and their DNA over time(Beerntsen,

40

James, Christensen 2000; Kartman 1953a; Ledesma and Harrington 2011). The current

study decapitated laboratory-infected mosquitoes to determine the number of mosquitoes

able to produce viable, emerging infective L3s to calculate infective rates. Afterwards,

molecular tests on the abdomens and head-thoraces were completed to determine the

extent to which the infection developed within females of each species, especially those

that were unable to produce viable infective L3s.

Aedes sierrensis. Molecular testing on samples of Aedes sierrensis elicited

consistently detectable, bright bands for D. immitis DNA. Although PCR is not a

quantitative test, band brightness should correlate with DNA template concentration.

Therefore, the bright bands observed likely demonstrate the presence of high levels of

DNA, or large quantities of the parasite. Across all three trials, 51.7% of abdomens

tested positive for D. immitis and 85% of those samples continued to produce infective

L3s. The few infective mosquitoes without positive abdomens were likely a result of

samples with low quantities of DNA that could not be consistently detected.

Aedes sierrensis has been considered the primary vector of D. immitis in northern

California since Weinmann and Garcia characterized its potential in 1974. By rearing

wild larvae from Marin County, CA and allowing the adults to feed on an infected dog,

they discovered that all surviving mosquitoes supported infective larvae under laboratory

conditions at 20 dpi(Weinmann and García 1974). To determine if this could be

replicated under more natural, ambient conditions, Walters and Lavoipierre infected wild

mosquitoes using a baited kennel in Tehama County, CA and maintained them at ambient

temperatures in an open laboratory(Walters and Lavoipierre 1982). In both rural and

residential areas they reported 100% infectivity. Similarly, our findings show that 55 to

41

100% of female Ae. sierrensis supported development of infective larvae by 21 dpi

between the three trials completed using the two strains at two different titers. Wild Ae.

sierrensis have been reported to support D. immitis development in the laboratory in

other Western states as well. Scoles et al. tested Ae. sierrensis in Utah to determine if

their arrival triggered the rise in D. immitis infections observed since 1987. They found

that 85% of mosquitoes supported infective L3 development by 15 dpi(Scoles, Dickson,

Blackmore 1993). The results may vary but the trend remains the same; Aedes sierrensis

demonstrates competence as a vector of D. immitis in the laboratory. Biologically, this

may be due to the lack of immunological responses or physiological barriers that inhibit

the development of the infection in other species(Ahid, Vasconcelos, Lourenço-de-

Oliveira 2000; Cancrini and Kramer 2001; Lowrie 1991; Nayar and Sauerman 1975;

Poinar and Leutenegger 1971). Additionally, this study found that raising the titer from

2.5 to 5 mff/μl significantly increased both the average number of L3s and the overall

infective rate. This builds on research by Lai et al. (2000) since it indicates some species

can support significantly more third-stage larvae relative to the microfilarial density. As

expected, this was met with a significant increase in mortality as increasing the filarial

burden within the mosquito further disrupts the vital excretory functions of the

Malpighian tubules(Palmer, Wittrock, Christensen 1986). Increasing the titer beyond this

point will likely continue to overwhelm the biology of Ae. sierrensis and produce even

fewer infective mosquitoes, confirming the hypothesis posed by Lai et al that suggested

lower microfilarial densities within definitive hosts likely contribute to a significant

portion of D. immitis transmission. Also, our data suggests a significant difference (χ2 =

5, df = 1, P = 0.02) in compatibility during the early stages of infection between the two

42

strains of D. immitis and Ae. sierrensis mosquitoes collected from Lake County, CA. The

infective rates for the Missouri strain were initially 0% at 15 dpi, while the Wildcat strain

presented with 53% at the same time point. By 21 dpi, however, the difference was no

longer significant.

In summary, Ae. sierrensis frequently produced positives for D. immitis DNA in

both abdomens and head-thoraces. In conjunction with high infective rates, Ae.

sierrensis has demonstrated its ability to support development of D. immitis to its

infective stage and likely plays a primary role in transmission in Lake County, CA, where

abundance is high. However, as this is not the case in some regions such as San Joaquin

County, CA, Ae. sierrensis likely plays a secondary role when abundance is low or its

seasonality does not overlap with conditions permissive for D. immitis development.

This, in part, could explain the 3.02% differential in D. immitis prevalence between the

two counties.

Culiseta incidens. Based on bloodmeal analysis studies, Cs. incidens has been

shown to feed frequently on domestic dogs. As such, we were very interested in

determining the potential of Cs. incidens as a vector in the laboratory(Theis et al. 2000).

Regrettably, the trial for this species was completed using the Missouri strain of D.

immitis as the source began to lose its patency. Furthermore, Cs. incidens were reluctant

to feed using our current methodology. Thus far, our only successful artificial feeding

method to date involved a dawn feeding using blood containing <1% sugar after roughly

108 hours of starvation. Results pertaining to this species were inconclusive as none of

the few mosquitoes that ingested the infected blood contained any sign of D. immitis,

whether it be infective larvae or positive molecular tests. Previous studies suggest that

43

Cs. incidens may be refractory to the development of larvae despite its willingness to

feed on domestic dogs, detection of D. immitis in wild-caught mosquitoes, and long

seasonality(Huang et al. 2013; Theis et al. 2000; Thiemann et al. 2012; Walters 1995).

Walters found that microfilariae failed to develop in 85% of Cs. incidens in Northern

California(Walters 1995). On the contrary, other studies from California have reported

that it can support D. immitis development and may serve as a vector of transmission. In

San Mateo County, CA, Acevedo (1982) reported that 7.14% (n=28) contained L3s either

in the Malpighian tubules or the proboscis by 16 and 21 dpi when allowed to feed on an

infected dog containing 11 mff/μl(Acevedo 1982). Similarly, Theis et al. infected reared

Cs. incidens from Southern California (Los Angeles County) and found that an average

of 0.4 L3s emerged from the 18 surviving mosquitoes dissected at 16 dpi(Theis et al.

2000). Additional trials are necessary to determine that status of Cs. incidens as a vector

in Lake County, CA.

Culex pipiens complex. In San Joaquin County, CA, Cx. pipiens complex is the

second most commonly trapped species, comprising 30.7% (n = 11,223) of collections in

2013 and accounting for 41.2% (n = 40/97) of all positive mosquito pools for D. immitis.

Seasonally, it is present during mid-March to November, the period in which 130 or

greater HDUs can be accumulated(Huang et al. 2013).

Molecular tests to detect D. immitis DNA in Cx. pipiens complex revealed

significantly fainter bands by comparison to Ae. sierrensis. To ascertain whether size or

quantity may cause negative molecular tests, samples of individual microfilaria and third

stage larvae were tested. While both were determined to be sufficient to elicit

consistently positive bands for D. immitis DNA, high densities of DNA other than that of

44

D. immitis could feasibly lower PCR efficiency and hinder detectability. Between the

two titers of the Missouri strain, 38% of abdomens tested positive for D. immitis and 64%

of those samples had a corresponding positive in the head-thorax. Midgut smears were

performed on both Cx. pipiens complex and Ae. sierrensis immediately post-feeding, but

only the latter contained microfilariae. The lack of microfilariae in the former was likely

biological in nature and specific to Cx. pipiens complex.

Of the many potential vectors under suspicion on the West Coast, Huang et al.

suggested that Culex pipiens complex (Culex pipiens and Culex quinquefasciatus) is

potentially one of the most important, at least in San Joaquin County, CA, due to its

spatiotemporal presence, abundance, and host feeding preference(Huang et al. 2013).

Lewandowski et al. came to this same conclusion, which prompted him to

experimentally infect reared adults from Lansing, Michigan using an infected

dog(Lewandowski Jr, Hooper, Newson 1980). Only a single infective larva was found in

a single mosquito at 15 dpi. Additionally, as microfilariae were unable to become

established in the Malpighian tubules in 87% of mosquitoes, they deduced that Cx.

pipiens was an inefficient host. In Lake County, CA, the potential of Cx. pipiens

complex may be limited due to its low relative abundance. In this study, infective rates

were never greater than 5%, regardless of the microfilarial titer used. This is consistent

with past studies that found Cx. pipiens complex to be refractory and prevent the

establishment of most worms within the Malpighian tubules(Hu 1931; Kartman 1953b;

Lewandowski Jr, Hooper, Newson 1980). Even when prevalence is as high as 37.1%,

which was the case in Rio de Janeiro, Brazil, Labarthe et al. found that microfilariae

rarely develops into infective L3s in Cx. pipiens complex. Of the 865 Cx.

45

quinquefasciatus mosquitoes collected, only 8 contained filariae at all, 3 of which ended

up supporting infective L3s(Labarthe et al. 1998). Based on these studies as well as the

lack of microfilariae observed in post-infected blood smears, it is possible that the

cibarial armature (located within the pharynx) may be responsible for the refractoriness

observed in Cx. pipiens complex. This explains why very few larvae are ever observed at

any individual stage within an individual mosquito since most worms never get a chance

to even become established in the first place(Lewandowski Jr, Hooper, Newson 1980).

Despite this, many authors agree that worms that can survive passage through the sharp,

sclerotized teeth of the cibarial armature find Cx. pipiens complex to be a favorable

vector(Cancrini and Kramer 2001). This seems to be the case for our study as well. For

example, if all the worms were shredded, then the residual D. immitis DNA contained

within the bloodmeal would have been digested prior to 15 dpi. While bloodmeal

digestion is temperature dependent, in most cases it shouldn’t take longer than a few

days(Jenkins 2004; MacDonald 1961). This was not the case in our trials, especially

considering that some mosquitoes developed infective L3s. While some worms certainly

survive the cibarial armature, the fact that most infected mosquitoes do not produce

infective larvae eludes to the presence of other biological or defensive mechanisms, such

as oxyhemoglobin and melanization(Ahid, Vasconcelos, Lourenço-de-Oliveira 2000;

Lowrie 1991; Nayar and Sauerman 1975; Poinar and Leutenegger 1971). Lowrie tested

two strains of Cx. quinquefasciatus (Leogane, Haiti & Convington, LA) at two different

titers (5 mff/μl & 20 mff/μl) using an artificial feeding apparatus and found that while up

to 27.5% of females supported infective stage larvae, oxyhemoglobin crystals were

capable of not only blocking and retaining microfilariae within the midgut, but also

46

damaging them which likely reduces the viability of the remaining worms that did

happen to survive and escape the midgut(Lowrie 1991). Similar to Ae. sierrensis, one

strain (Haiti) consistently produced more infective larvae. This study is another example

of just how much intraspecific diversity exists even amongst seemingly refractory

species. Unlike Ae. sierrensis, increasing the titer did not result in additional infective

mosquitoes or a higher mean number of L3s. One possibility is that centrifugation may

have altered the viability of the infected blood by damaging the worms or altering the

consistency of the blood. Blood that coagulates too quickly may prevent microfilariae

from escaping the blood bolus and reaching the Malpighian tubules before the meal is

digested(Cancrini and Kramer 2001). An additional repeat may be necessary to

determine if one of these factors may have contributed to the low levels of infectivity

observed. Additionally, some literature speculates that in regions where D. immitis is

hyperendemic, transmission may occur at significantly higher rates(Cancrini and Kramer

2001; Genchi, Kramer, Prieto 2001).

Culex pipiens complex is one of the most abundant mosquitoes in San Joaquin

County, CA. While Cx. pipiens complex possesses all the fundamental traits of a

competent vector, such as frequent D. immitis field detections, ecological overlap, and

appropriate blood feeding patterns, our results suggest that Cx. pipiens complex is

relatively refractory at low microfilarial densities equal to or below 5 mff/μl(Huang et al.

2013; Thiemann et al. 2012). Furthermore, some members of Cx. pipiens complex (Cx.

pipiens, not Cx. quinquefasciatus) are autogenous, meaning that they ingest fewer

bloodmeals throughout their lifetime and are less likely to acquire or transmit an

infection(Cancrini and Kramer 2001). Regardless, Cx. pipiens complex can support

47

complete development in some instances and should be regarded as a competent vector.

Whether Cx. pipiens complex is a primary or secondary vector of importance depends on

how abundant it may be in any given area.

Conclusion. In summary, Ae. sierrensis, Cx. pipiens complex, and Cs. incidens

demonstrated that additional information was necessary to evaluate their competence in

the laboratory and determine their potential as vectors of D. immitis. Based on our

findings, only Ae. sierrensis and Cx. pipiens complex have been confirmed as vectors of

D. immitis as they were able to support infective L3 development. Both species should

be targeted in Northern California to reduce the incidence of new cases of canine

heartworm disease. Even a species of low competence such as Cx. pipiens complex can

result in a noticeable degree of activity if abundance is high, which may be the case in

San Joaquin County, CA. Conversely, Ae. sierrensis, which has a low abundance and

short seasonal activity in San Joaquin County, CA may cause a significant number of

cases if practically all infected females can transmit the parasite after being infected. In

Lake County, CA, these concerns are heightened as these limitations are not a concern

and Ae. sierrensis likely plays a primary role in transmission. While Cx. pipiens complex

is present here as well, less risk of transmission is associated in this case due to

competence and relative abundance. In the future, we would like to complete our

assessment of Cs. incidens as well as study other potential vectors such as Cx. tarsalis.

Other avenues worth investigating include the effects of coinfection and the clarification

of the life cycle of D. immitis as many papers gloss over the finer details of development.

Although this study serves as a foundation, additional research using both local strains of

D. immitis and potential mosquito vectors may be necessary to characterize local

48

transmission due to the diversity that exists within populations of both mosquitoes and D.

immitis alike.

49

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