Molecular Characterization of Adult Diapause in the Northern House Mosquito, Culex Pipiens

Home , Culex pipiens, Embryonic diapause, Hibernaculum (zoology), Hibernation

MOLECULAR CHARACTERIZATION OF ADULT DIAPAUSE IN THE NORTHERN HOUSE MOSQUITO, CULEX PIPIENS

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

Rebecca M. Robich, M.S.

*****

The Ohio State University 2005

Dissertation Committee:

Professor David L. Denlinger, Advisor Approved by

Professor Donald H. Dean ______Professor Glen R. Needham Advisor Graduate Program in Entomology Professor Brian H. Smith

ABSTRACT

In the northern United States, Culex pipiens (L.), a major avian vector of several

arthropod-borne viruses, spends a good portion of the year in a state of developmental

arrest (diapause). Although the physiological and hormonal aspects of Cx. pipiens

diapause have been well-documented, there is little known on the molecular aspects of

this important stage. Using suppressive subtractive hybridization (SSH), 40 genes

differentially expressed in diapause were identified and their expression profiles were

probed by northern blot hybridization. These genes have been classified into 8 distinct

groupings: regulatory function, food utilization, stress response, metabolic function,

cytoskeletal, ribosomal, transposable elements, and genes with unknown functions.

Among 32 genes confirmed by northern blot hybridization, 6 are upregulated specifically in early diapause, 17 are upregulated in late diapause, and 2 are upregulated

throughout diapause. In addition, 2 genes are diapause downregulated and 5 remained

unchanged during diapause. Two regulatory genes upregulated in late diapause,

ribosomal protein (rp) S3A and rpS6, are of particular interest for their potential

involvement in developmental arrest. In other mosquito species, these genes are

upregulated prior to oogenesis, and their suppression leads to a disruption in ovarian

development. Since arrested ovarian development is a key characteristic of Cx. pipiens

diapause, the lack of expression in early diapause may be key to this developmental

ii arrest. Of equal interest is the upregulation of two stress-response genes in late diapause.

The molecular chaperone, heat shock protein 23 (hsp23) is slightly upregulated in late

diapause and may be involved in protecting females from environmental stresses. In

addition, a gene encoding an enzyme involved in oxidizing certain insecticides in

insecticide-resistant strains of Cx. quinquefasciatus, aldehyde oxidase, is strongly upregulated in Cx. pipiens late diapause.

SSH and northern blot analysis also demonstrated the strong upregulation of a

muscle-specific actin gene in Cx. pipiens early diapause. Although the upregulation of

this cytoskeletal gene in early diapause may seem counterintuitive, females actively fly

during diapause preparation in search of sugar meals and a protective site for

overwintering. In addition, two ribosomal genes (large ribosomal subunit L18 and large

ribosomal subunit) are upregulated in early diapause suggesting that their function is

restricted to diapause preparation. Curiously, two genes encoding transposable elements,

transposon T1-2 and Mimo-Cp2, are upregulated during late diapause in Cx. pipiens, but

the function of these genes during diapause is unknown. Nine genes were also isolated

by SSH with unknown identities, and all but one are upregulated in late diapause, as

identified by northern blot hybridization

In addition to the aforementioned genes, two genes encoding the blood digestive

enzymes, trypsin and chymotrypsin-like, are downregulated in early diapause, and

concurrently a gene associated with the accumulation of lipids, fatty acid synthase, is

highly upregulated. As females enter diapause, fatty-acid synthase is only sporadically

expressed, while the expression of trypsin and chymotrypsin-like remain undetectable

iii until late diapause, when females prepare to take a blood meal upon diapause break. This

is the first molecular evidence demonstrating that diapause in Cx. pipiens evokes a

molecular switch from blood-feeding in nondiapausing individuals to sugar feeding in

diapause-destined females.

The increase in sugar feeding in diapause-destined Cx. pipiens and subsequent

accumulation of lipid reserves likely requires a high amount of energy. This may be

reflected in the upregulation of genes encoding two respiratory enzymes, cytochrome c

oxidase subunit I (COI) and cytochrome c oxidase subunit III (COIII), during diapause

preparation. Although transcript levels decline in mid-diapause when females enter an

inactive state, levels again rise in late diapause, just prior to diapause break. There are no differences in mtDNA levels between nondiapausing and diapausing Cx. pipiens, thus suggesting that mitochondrial numbers are not reduced during diapause. Regulation of

COI and COIII is thus likely to be under transcriptional control.

In addition to genes obtained through SSH, heat shock protein 70 was isolated

from Cx. pipiens by 5- and 3- rapid amplification of cDNA ends. This gene is upregulated upon heat shock and recovery from cold shock in diapausing and nondiapausing females, but is not upregulated as a component of the diapause program.

However, diapausing females reared at 18°C survive cold exposure (-5°C) nearly twice as long as its nondiapausing counterparts reared at 18°C and 10 times as long as nondiapausing mosquitoes reared at 25°C. Diapausing females are also more desiccation resistant (1.6 to 2 fold) than nondiapausing females, regardless of rearing temperature.

iv These results may be useful when comparing the molecular aspects of diapause across different taxa and developmental stages. In addition, this work may be helpful in understanding the transseasonal maintenance of viruses that utilize overwintering insects.

For Michael

ACKNOWLEDGMENTS

I am grateful to my advisor, David L. Denlinger, Ph.D., for his intellectual guidance and continued support for this project, and for his assistance in editing each chapter.

I would like to thank my committee members, Drs. Donald H. Dean, Glen R. Needham, and Brian H. Smith, for their ideas and comments in bettering this thesis.

I am also grateful for the assistance of Joseph P. Rinehart, Ph.D., and Linda Kitchen in helping to make this dissertation come together.

I would like to thank those involved in the surveillance of West Nile virus in overwintering mosquitoes, including Richard Gary and Robert A. Restifo of the Ohio Department of Health's Vector-Borne Disease Program, and Joseph Lynch of the Cuyahoga County Board of Health.

I appreciate the efforts of Woodbridge Foster, Ph.D., in helping to establish the Culex pipiens (Buckeye strain) colony, who collected the original larvae from his backyard in September 2000.

vii

VITA

September 20, 1974 .…...... Born – Painesville, Ohio

1992 ..…………………..… Bachelor of Science, Environmental Biology Ohio University

1997-1998 .………………. Graduate Teaching Associate Ohio University

1998 .……………………... Master of Science, Environmental Studies Ohio University

1999-2000 .………………. Pre-Doctoral Fellow Virology Division U.S. Army Medical Research Institute of Infectious Diseases

2000-2004 .………………. Graduate Teaching and Research Associate The Ohio State University

2001 .……………………... Medical Entomology Summer Intern Ohio Department of Health

PUBLICATIONS

Moll, R.M., Romoser, W.S., Modrzakowski, M.C, Moncayo, A.C., and Lerdthusnee, K. 2001. Meconial peritrophic membranes and the fate of midgut bacteria during mosquito (Diptera: Culicidae) metamorphosis. Journal of Medical Entomology 38:29-32.

Romoser, W.S., Moll, R.M., Moncayo, A.C., and Lerdthusnee, K. 2000. The occurrence and fate of the meconium and meconial peritrophic membranes in pupal and adult mosquitoes (Diptera: Culicidae). Journal of Medical Entomology 37:893-6.

FIELDS OF STUDY

Major Field: Entomology viii

TABLE OF CONTENTS

Abstract ………………………………………………………………………………….. ii

Dedication ………………………………………………………………………………. vi

Acknowledgements …………………………………………………………………...... vii

Vita ……………………………………………………………………………………. viii

List of Tables …………………………………………………………………………… xi

List of Figures ………………………………………………………………………….. xii

Chapters:

1. Introduction ……………………………………………………………………… 1 References ……………………………………………………………… 12

2. Diapause-specific gene expression in adults of the northern house mosquito, Culex pipiens L., identified by suppressive subtractive hybridization. Abstract ………………………………………………………………… 16 Introduction …………………………………………………………….. 17 Materials and Methods …………………………………………………. 19 Results ………………………………………………………………….. 23 Discussion ……………………………………………………………… 27 References ……………………………………………………………… 38

3. Diapause in the mosquito Culex pipiens evokes a metabolic switch that shuts down genes encoding blood digestive enzymes and upregulates a gene associated with sugar utilization and lipid storage.

Abstract ………………………………………………………………… 49 Introduction …………………………………………………………….. 50 Materials and Methods …………………………………………….…… 52 Results ……………………………………………………………….…. 57 Discussion ……………………………………………………………… 63 References ……………………………………………………………… 69

4. Downregulation of mitochondrial mRNA expression, but not mitochondrial number, during adult diapause in the northern house mosquito, Culex pipiens.

Abstract ………………………………………………………………… 82 Introduction ………………………………………………………….…. 83 Materials and Methods ……………………………………………….… 85 Results ………………………………………………………………….. 90 Discussion ……………………………………………………………… 94 References ……………………………………………………………… 99

5. Enhanced cold and desiccation tolerance in diapausing adults of Culex pipiens, and a role for hsp70 in response to cold shock but not as a component of the diapause program.

Abstract ……………………………………………………………..… 109 Introduction …………………………………………………………… 110 Materials and Methods ………………………………………………... 112 Results ………………………………………………………………… 120 Discussion …………………………………………………………….. 126 References ………………………………………………………….…. 132

Conclusions …………………………………………………………………………… 142

Appendix ……………………………………………………………………………… 149

Materials and Methods ……………………………………………...… 150 Results and Discussion ……………………………………………..… 155 References …………………………………………………………..… 159

Bibliography ………………………………………………………………………….. 149

LIST OF TABLES

Table Page

2.1 Diapause upregulated genes from Cx. pipiens, isolated by suppressive subtractive hybridization ………………………………………..… 44

2.2 Diapause downregulated genes from Cx. pipiens, isolated by suppressive subtractive hybridization ………………………………………….. 46

A.1 Ratio and percent of overwintering Cx. pipiens moving within Their hibernation sites from January to April, 2004 ………………………..… 170

LIST OF FIGURES

Figure Page

2.1 Northern blot hybridization of Cx. pipiens diapause upregulated SSH clones … 47

2.2 Northern blot hybridization of Cx. pipiens diapause downregulated SSH clones …………………………………………………………….……….. 48

3.1 Nucleotide and deduced amino acid sequences of Cx. pipiens trypsin cDNA … 73

3.2 Nucleotide and deduced amino acid sequences of Cx. pipiens chymotrypsin-like serine protease ……………………………………………... 74

3.3 Nucleotide and deduced amino acid sequences of Cx. pipiens fatty acid synthase cDNA …………………………………………………….... 75

3.4 Multiple sequence alignment of the deduced Cx. pipiens trypsin with other insect trypsins retrieved from GenBank ..…………………………... 76

3.5 Multiple sequence alignment of the deduced Cx. pipiens chymotrypsin-like serine protease with other insect serine proteases retrieved from GenBank .….. 77

3.6 Multiple sequence alignment of the deduced Cx. pipiens fatty acid synthase with other insect fatty acid synthases retrieved from GenBank ...……………... 78

3.7 Northern blot hybridization of diapause-regulated genes involved in blood meal vs. sugar meal digestion in Cx. pipiens ……………………………. 79

3.8 Temporal pattern of expression of the genes encoding the digestive enzymes trypsin, chymotrypsin-like, and fatty acid synthase ……………………………. 80

3.9 Expression of trypsin, chymotrypsin-like, and fatty acid synthase throughout diapause and when diapause is broken …………………………….. 81

4.1 Nucleotide sequence and deduced amino acid sequence of Cx. pipiens cytochrome c oxidase subunit I cDNA …………………………... 102

xii 4.2 Nucleotide sequence and deduced amino acid sequence of Cx. pipiens cytochrome c oxidase subunit III cDNA ……………………….... 104

4.3 Multiple sequence alignment of the deduced Cx. pipiens cytochrome c oxidase subunit I (COI) with other insect COI sequences retrieved from GenBank ….. 105

4.4 Multiple sequence alignment of the deduced Cx. pipiens cytochrome c oxidase subunit III (COIII) with other insect COIII sequences retrieved from GenBank ……………………………………. 106

4.5 Temporal pattern of expression of the genes encoding the mitochondrial respiratory enzymes COI and COIII ………………………………………….. 107

4.6 Expression of COI and COIII throughout diapause and when diapause is broken ……………………………………………………………. 108

5.1 Percent survival (mean + SE) of Cx. pipiens females after exposure to different durations of low temperature (-5°C) ……………...……………… 136

5.2 Percent survival (mean + SE) of Cx. pipiens females at various relative humidities ……………………..…………...……………… 137

5.3 Complete nucleotide sequence and deduced amino acid sequences of Cx. pipiens hsp70 cDNA …………………………………………………... 138

5.4 Multiple sequence alignment of the deduced Cx. pipiens hsp70 with other hsp70 sequences retrieved from GenBank ………………………... 140

5.5 Northern blots showing expression of heat shock protein 70 in Cx. pipiens females at different times (hours) following a 4 h exposure to –5°C ………… 141

A.1 Diagrams and photos of three Ohio culverts …………………………………. 161

A.2 Average temperature recorded in three Ohio culverts from November 19, 2003 through April 30, 2004, at hourly intervals ……………... 164

A.3 Average relative humidity recorded in three Ohio culverts from November 19, 2003 through April 30, 2004, at hourly intervals ……………... 167

xiii

CHAPTER 1

INTRODUCTION

Many important mosquito vectors of transmissible disease that live in temperate

zones enter an overwintering dormancy (diapause). Though the period of dormancy

results in a significant decrease or absence of vector-borne diseases during this portion of

the year, the overwintering stage of the mosquito can harbor pathological agents during

this time and thus reinitiate disease transmission the following spring. This appears to be

a likely scenario that may have contributed to the reappearance of West Nile virus in the

New York City area following the initial outbreak in the summer of 1999 (Nasci et al.,

2001). In this case, diapausing adult females of Culex pipiens were found to harbor the

virus during the winter months, and such mosquitoes serve as one possible mechanism

for reintroducing the disease into bird populations the following season. More recently,

West Nile virus was noted in overwintering populations of Cx. pipiens collected during

February 2003 in spring houses in eastern Pennsylvania (Kristen Bardell, Pa. Dept. of

Environmental Protection, personal communication) and from populations collected in

2004 from culverts in Boston (Andrew Spielman, Harvard University, personal communication).

1 The diapausing stage thus emerges as an extremely important stage for

understanding certain disease transmission cycles. Not only is this stage critical for

understanding the seasonal occurrence of vector-borne diseases, but understanding the physiological and molecular basis for diapause could provide significant clues for understanding how the pathogen is regulated. The focus of this work is on the molecular mechanisms that serve to regulate the adult reproductive diapause in the northern house mosquito Cx. pipiens, the primary sylvatic vector of West Nile virus. Many aspects of diapause in this species have been well documented. There is a good database that describes the physiological features of this diapause, its environmental regulators, and the hormonal control mechanism, but what is currently lacking is an understanding of its molecular underpinning.

Diapause in Cx. pipiens

The northern house mosquito, Cx. pipiens L., is a member of the Cx. pipiens complex, which was once considered to be comprised of a number of different subspecies

(Mattingly et al., 1951). In the United States, the complex includes the southern house mosquito, Cx. quinquefasciatus Say, and two variants of the northern house mosquito: an autogenous form, Cx. pipiens var. molestus, which can produce an initial batch of eggs without a blood meal, and an anautogenous form, Cx. pipiens pipiens, which requires a blood meal for egg production. Both autogenous and anautogenous variants of Cx. pipiens exist north of 39o spanning across the temperate zones of North America, Europe, and parts of Asia (Barr, 1957), but it is only the anautogenous form (requires a blood

2 meal for egg production) that overwinters in an adult reproductive diapause (Spielman,

1964; 1971; Spielman and Wong, 1973a). Although males are occasionally found in hibernation sites, it is only the females that enter diapause and successfully overwinter.

Mating occurs prior to the entry into diapause, and diapausing females are rarely found without sperm (Spielman, 1964).

Environmental Regulation of Diapause in Cx. pipiens:

Like diapause in most temperate zone species, short daylength and low temperature are the primary environmental factors dictating the entry into diapause

(Eldridge, 1966; Sanburg and Larsen, 1973; Spielman and Wong, 1973b). Cx. pipiens reared under <12 h light per day and at temperatures below 20°C results in >80% diapausing females, while those reared at longer photoperiods and low temperature

(<20°C) leads to fewer than 45% of females in diapause (Spielman and Wong, 1973b.

Rearing females at 25°C or higher, however, produces all nondiapausing females, regardless of photoperiod (Eldridge, 1966; Spielman and Wong, 1973b; Sanburg and

Larsen, 1973). Thus, diapause in Cx. pipiens is determined by the combined effects of temperature and photoperiod.

The photosensitive stage used for programming diapause begins during late larval life and persists into the early days of adult life (Oda, 1968; Sandburg and Larsen, 1973;

Spielman and Wong, 1973b), but the period of maximum sensitivity occurs shortly after the larval-pupal molt (Spielman and Wong, 1973b). In the late summer and early fall, females that receive diapause-inducing conditions during the photosensitive stage

3 undergo a number of behavioral and physiological changes as adults; diapause-destined

females show a boost in sugar feeding, blood feeding ceases due to a lack of host-seeking

response, their fat body hypertrophies, and they seek well-protected sites for hibernation.

The Preparatory Phase of Cx. pipiens Diapause

In association with the entry into diapause, females increase their consumption of carbohydrates found in plant sources such as nectar and rotting fruits, which leads to a hypertrophy of their fat body (Mitchell and Briegel, 1989a; Bowen, 1992). Diapause- destined females feed more readily on sugar and for a longer time than their nondiapausing counterparts during the first few weeks of adult life (Bowen, 1992), which leads to the accumulation of nearly twice as many lipid reserves (Mitchell and Briegel,

1989a). These lipid reserves are gradually depleted during the course of winter; laboratory and field studies indicate fat stores are depleted by 80% or more over a 5 month period (Onyeka and Boreham, 1987; Mitchell and Briegel, 1989a)

Another key characteristic of prehibernating females is an absence in host-seeking behavior, which results in a lack of blood feeding at this time (Mitchell, 1983; Mitchell and Briegel, 1989a; Bowen, 1992). Whether or not diapause-destined females will take a blood meal prior to entering hibernation has been a great debate in the literature. Females placed in close proximity to a host can be enticed to take a blood meal (Eldridge, 1966;

Eldridge and Bailey, 1979; Mitchell, 1983), but this bypasses the host-seeking step necessary under natural conditions. When laboratory-reared females are offered a blood meal in large containers where host-seeking is essential, only a small percentage will

4 actually take a blood meal (Mitchell, 1983). Bowen (1990) demonstrated that Cx. pipiens reared under diapause-inducing conditions lack high-sensitivity olfactory responsiveness to the host-attractant, lactic acid. Furthermore, force-fed diapausing Cx. pipiens eject most of the blood ingested within 24 h of taking a blood meal, and any remaining blood is incompletely digested and is not used to increase lipid reserves or to initiate vitellogenesis (Mitchell and Briegel, 1989b). In addition, winter-collected females show no signs of blood-feeding, and only an occasional blood-fed female can be found resting in sites after April, once diapause has been terminated (Onyeka and Boreham, 1987).

After the acquisition of sufficient lipid reserves for winter survival, Cx. pipiens seek well-protected sites for hibernation: caves, hollows, and various artificial shelters including culverts and unheated basements are commonly selected (Vinogradova, 2000).

Cx. pipiens do best in sites that remain above 0°C with a small fluctuation in temperature around 1-2°C per month, and have a high relative humidity over 90% (Minar and Ryba,

1971). In the Boston area, females first begin appearing in such sites in mid-August and reach peak density by early October (Spielman and Wong, 1973a). Similar patterns have been noted in England (Onyeka and Boreham, 1987). Females are active in their sites throughout the winter; considerable movement of diapausing Cx. pipiens was observed in each month tested from September to April in mosquitoes overwintering in southern

England (Onyeka and Boreham, 1987).

5 Arrest in Ovarian Development

Once in diapause, the female’s follicles are arrested at Christophers’ Stage I or

perhaps slightly earlier (Oda, 1968; Sanburg and Larsen, 1973; Spielman and Wong,

1973b). At this stage, the follicles contain distinct oocytes surrounded by a follicular

epithelium, but no lipid droplets are yet present in the ooplasm (Clements, 1992). The

most advanced follicles in diapausing females measure 40-50 µm in length, a length that

is no more than 1.5 x that of the germarium (Spielman and Wong, 1973b). In an experimental simulation of diapause, follicle size remained at Christophers’ Stage I for

the first 10 weeks and then gradually increased to Stage II by 22 weeks (Readio et al.,

1999).

Only parous females can be collected by host-attractant CO2 traps in the early fall,

while females collected from overwintering sites at this time are all nulliparous (Andrew

Spielman, personal communication). This suggests that females emerging after August that have follicles in arrested development do not seek a blood meal for further development at this time. Females collected in CO2 traps are older mosquitoes and will

likely produce diapause-destined progeny if a blood meal is available.

Hormonal Regulation of Diapause in Cx. pipiens

As is the case of most adult diapauses (Denlinger, 1985; 2002), the diapause of

Cx. pipiens appears to be initiated by a shut-down in the production of juvenile hormone

(JH) by the corpora allata (Spielman, 1974). During the first hours of adult life, there is

no production of juvenile hormone in either nondiapausing or diapausing females, but by

6 24 h nondiapausing females produce 10 times as much juvenile hormone as their

diapausing counterparts (Readio et al., 1999). Four weeks into diapause, juvenile

hormone titers increase slightly and plateau at this level until week 12; after this time

there is a gradual increase in JH production until levels comparable to 3-day old

nondiapausing females are reached by week 20 (Readio et al., 1999). This slow rise in

JH corresponds to the gradual increase in follicle size also seen in diapausing Cx. pipiens

throughout the winter period (Readio et al., 1999).

Moving diapausing females to nondiapause conditions (long daylength and high temperature) results in an increase in JH titers over a 5 day period (Readio et al., 1999)

In addition, allatectomized females of Cx. pipiens that were not programmed for diapause

enter a diapause-like state and cease host-seeking behavior (Meola and Petralia, 1980),

and diapausing females can be prompted to reinitiate ovarian development by topical

application of JH-III or by application of JH analogs (Spielman, 1974; Readio and Meola,

1985; Readio et al., 1988). All of this evidence points to this diapause being a classic

case of regulation by JH.

Termination of Diapause

Few Cx. pipiens can be found resting in overwintering sites after April, and

females have been observed to leave their sites as early as March (Minar and Ryba, 1971;

Onyeka and Boreham, 1987). The termination of diapause is dependent primarily on

temperature (Oktyabrskaya et al., 1965); females departed from a warm site in Moscow

nearly a month before females departed from a nearby, cooler site. In field-collected Cx.

7 pipiens from early winter, nearly 2 weeks of exposure to long-day length was required before diapause was broken, whereas a few days to less than a week of long-day conditions was required to terminate diapause in females collected from overwintering sites in March and April (Onyeka and Boreham, 1987). This suggests that females gradually come out of diapause through the course of winter and are prepared to resume nondiapausing activities once suitable environmental conditions are attained.

Transeasonal Maintenance of West Nile Virus

The failure to find evidence for blood feeding in overwintering Cx. pipiens suggests that a blood meal is an unlikely source of West Nile virus, and thus vertical transmission is the most likely scenario. Cx. pipiens is the primary sylvatic vector of

West Nile virus in the northern United States. For example, from 6,900 pools of mosquitoes tested for West Nile virus in the summer of 2002 in Ohio, 1,600 were positive by PCR, and 75% of these were identified as Cx. spp. ( pipiens or restuans)

(Richard Gary, Ohio Department of Health, personal communication). Laboratory experiments suggest that Cx. pipiens is a poor vector of West Nile virus since only 2% of orally infected mosquitoes obtained disseminated infections; however, 88% of those that became infected were able to subsequently transmit the virus to a naïve chicken (Turell et al., 2001).

In the laboratory, Cx. pipiens intrathoracically inoculated with West Nile virus showed low vertical transmission rates of <0.3%, which corresponds to a minimal filial infection rate ~ 1.8 (Dohm et al., 2002). Intrathoracic inoculation is not a natural means

8 of acquiring virus, and these numbers likely under-represent what happens in the field.

Although photoperiod has not been evaluated as a factor contributing to the ability of Cx.

pipiens to acquire and transmit West Nile virus, environmental temperature has been

tested and appears to play a role in the ability to recover live virus from orally-infected

females (Dohm and Turell, 2001). Live West Nile virus was recovered from 100% of

infected Cx. pipiens held at a high temperature (26°C), whereas no live virus was

recovered from mosquitoes held at 10°C (Dohm and Turell, 2001).

Molecular Regulation of Mosquito Diapause

The molecular characteristics of diapause in Cx. pipiens have not been explored.

Indeed, very little is known of these events in any mosquito vector, although such work has been initiated on the embryonic diapause of Oclerotatus triseriatus, an important vector of La Crosse encephalitis (Blitvich et al., 2002). Thus far, these workers have identified mRNA sequences present during embryonic diapause of Oc. triseriatus, using primers designed to amplify sequences that the La Crosse virus cap scavenges. Among cDNA fragments that have been identified are a mitochondrial cytochrome c oxidase subunit, 18S and 28S ribosomal RNAs, protein disulfide-isomerase, guanine nucleotide- binding protein, human N33 protein, and several novel transcripts. It is still too early to know if any of these genes are indeed involved in regulation of this diapause or in the regulation of viral transcription. Embryonic and reproductive diapauses are sufficiently

9 different in terms of hormonal regulation (Denlinger, 1985; 2002), thus it is not at all

certain whether the same or similar molecular mechanisms operate in diapauses of

different stages.

From our laboratory studies with the pupal diapause of flesh flies it is evident that

the expression of many genes is shut down during diapause, while a small cluster of

genes (approximately 4 %) are diapause upregulated (Denlinger, 2002). Several classes

of diapause upregulated genes have been noted. Some, such as those that encode heat shock protein (hsp) 23 and hsp70, are upregulated throughout diapause (Yocum et al.,

1998; Rinehart et al., 2000). Another group, represented by cystatin, is expressed only in early diapause and may be involved in initiating the arrest in development (Goto and

Denlinger, 2002). Yet another category, represented by ultraspiracle, is upregulated only in late diapause and is likely to be involved in the processes leading to diapause termination (Rinehart et al., 2001). Of potentially equal importance are key regulatory genes that are shut down during diapause. In flesh flies, one such gene encodes proliferating cell nuclear antigen, a key regulator of the cell cycle (Tammariello and

Denlinger, 1998). A shut down in expression of this gene is possibly responsible for the cell cycle arrest that characterizes pupal diapause in flesh flies. Experiments currently

underway with this system focus on a search for additional diapause-regulated genes and

the functions of those genes in relation to diapause.

Molecular studies on adult diapauses, such as that noted in Cx. pipiens, have thus far received little attention. The adult diapause of the Colorado potato beetle is arguably the best-understood adult diapause, but little work has focused on molecular aspects of

10 this diapause. The first paper of this nature (Yocum, 2001) documents the upregulation of hsp70 during adult diapause in the Colorado potato beetle, an intriguing result suggesting that there may indeed be some parallels between the molecular events regulating diapause in different species and stages.

Research Foci

This study investigates the molecular mechanisms regulating adult reproductive

diapause of Cx. pipiens, and also examines responses to environmental stress such as

temperature and relative humidity in the field and laboratory settings. Suppressive

subtractive hybridization was used to identify genes differentially expressed in early and

mid diapause, and expression of these genes was confirmed by northern blot

hybridization. From this, 9 categories of genes have been identified as either upregulated

or downregulated during diapause, and these include genes involved in regulatory

functions, genes associated with food utilization, stress-response genes, genes of

metabolic function, cytoskeletal genes, those of mitochondrial origin, genes encoding

ribosomal proteins, transposable elements, and genes with unknown function.

This research will provide a basis for understanding the molecular regulation of

diapause in this important species and may prove valuable for probing potential

commonality in the diapauses of different taxa and developmental stages. In addition,

these results may also be useful for understanding how the replication of West Nile virus

within Cx. pipiens can be seasonally shut down in the autumn and again reinitiated the

following spring.

REFERENCES

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Blitvich, B.J., Rayms-Keller, A., Blair, C.D., and Beaty, B.J. 2001. Identification and sequence determination of mRNAs detected in dormant (diapausing) Aedes triseriatus mosquito embryos. DNA Sequence 12:197-202.

Bowen, M.F. 1990. Post-diapause sensory responsiveness in Culex pipiens. Journal of Insect Physiology 36:923-929.

Bowen, M.F. 1992. Patterns of sugar feeding in diapausing and nondiapausing Culex pipiens (Dipetera: Culicidae) females. Journal of Medical Entomology 29:843-849.

Bowen, M.F., Davis, E.E., and Haggar, D.A. 1988. A behavioral and sensory analysis of host-seeking behavior in the diapausing mosquito Culex pipiens. Journal of Insect Physiology 34:805-813.

Clements, A.N. 1992. The biology of mosquitoes, volume I: development, nutrition, and reproduction. CABI Publishing, Cambridge, pp. 340-359.

Denlinger, D.L. 1985. Hormonal control of diapause. In: Kerkut, G.A., Gilbert, L.I. (Eds.), Comprehensive insect physiology, biochemistry and pharmacology, Vol. 8. Pergamon Press, Oxford, pp. 353-412.

Denlinger, D.L. 2002. Regulation of diapause. Annual Review of Entomology 47:93- 122.

Dohm, D.J., Sardelis, M.R., and Turell, M.J. 2002. Experimental transmission of West Nile virus by Culex pipiens (Diptera: Culicidae). Journal of Medical Entomology 39:640-644.

Dohm, D.J., and Turell, M.J. 2001. Effect of incubation at overwintering temperatures on the replication of West Nile virus in New York Culex pipiens (Diptera: Culicidae). Journal of Medical Entomology 38:462-464.

Eldridge, B.F. 1966. Environmental control of ovarian development in mosquitoes of the Culex pipiens complex. Science 151:826-828.

Eldridge, B.F., and Bailey, C.L. 1979. Experimental hibernation studies on Culex pipiens (Diptera: Culicidae): reactivation of ovarian development and blood-feeding in prehibernating females. Journal of Medical Entomology 15:462-467.

Goto, S.G., and Denlinger, D.L. 2002. Genes encoding two cystatins in the flesh fly Sarcophaga crassipalpis and their distinct expression patterns in relation to pupal diapause. Gene 292:121-127.

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Meola, R.W., and Petralia, R.S. 1980. Juvenile hormone induction of biting behaviour in Culex mosquitoes. Science 209:1548-1550.

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Mitchell, C.J. 1983. Differentiation of host-seeking behavior from blood-feeding behavior in overwintering Culex pipiens (Diptera: Culicidae) and observations on gonotrophic dissociation. Journal of Medical Entomology 20:157-163.

Mitchell, C.J., and Briegel, H. 1989a. Inability of diapausing Culex pipiens (Diptera: Culicidae) to use blood for producing lipid reserves for overwinter survival. Journal of Medical Entomology 26:318-326.

Mitchell, C.J., and Briegel, H. 1989b. Fate of the blood meal in force-fed, diapausing Culex pipiens (Diptera: Culicidae). Journal of Medical Entomology 26:332-341.

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Oda, T. 1968. Studies on the follicular development and overwintering of the house mosquito, Culex pipiens pallens in Nagasaki area. Tropical Medicine 10:195-216.

Oktyabrskaya, T.A., Astakhova, N.A., and Boiko, L.P. 1965. Materials on the species composition, biology and ecology of mosquitoes observed near Moscow. Meditsinskaia Parazitologiia i Parazitarnye Bolezni 34:510-513.

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13 Readio, J., Chen, M., and Meola, R. 1999. Juvenile hormone biosynthesis in diapausing and nondiapausing Culex pipiens (Diptera: Culicidae). Journal of Medical Entomology 36:355-360.

Rinehart, J.P., Cikra-Ireland, R.A., Flannagan, R.D., and Denlinger, D.L. 2001. Expression of ecdysone receptor is unaffected by pupal diapause in the flesh fly, Sarcophaga crassipalpis, while its dimerization partner, USP, is downregulated. Journal of Insect Physiology 47:915-921.

Rinehart, J.P., Yocum, G.D., and Denlinger, D.L. 2000. Developmental upregulation of inducible hsp70 transcripts, but not the cognate form, during pupal diapause in the flesh fly, Sarcophaga crassipalpis. Insect Biochemistry and Molecular Biology 30:515-521.

Sanburg, L.L., and Larsen, J.R. 1973. Effect of photoperiod and temperature on ovarian development in Culex pipiens pipiens. Journal of Insect Physiology 19:1173-1190.

Spielman, A. 1964. Studies on autogeny in Culex pipiens populations in nature. I. Reproductive isolation between autogenous and anautogenous populations. American Journal of Hygiene 80:175-183.

Spielman, A. 1971. Studies on autogeny in natural populations of Culex pipiens. II. Seasonal abundance of autogenous and anautogenous populations. Journal of Medical Entomology 8:555-561.

Spielman, A. 1974. Effect of synthetic juvenile hormone on ovarian diapause of Culex pipiens mosquitoes. Journal of Medical Entomology 11:223-225.

Spielman, A., and Wong, J. 1973a. Studies on autogeny in natural populations of Culex pipiens. III. Midsummer preparation for hibernation in anautogenous populations. Journal of Medical Entomology 10:319-324.

Spielman, A., and Wong, J. 1973b. Environmental control of ovarian diapause in Culex pipiens. Annals of the Entomological Society of America 66:905-907.

Tammariello, S.P., and Denlinger, D.L. 1998. G0/G1 cell cycle arrest in the brain of Sarcophaga crassipalpis during pupal diapause and the expression pattern of the cell cycle regulator, proliferating cell nuclear antigen. Insect Biochemistry and Molecular Biology 28:83-89.

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14 Vinogradova, E.B. 2000. Culex pipiens pipiens mosquitoes: taxonomy, distribution, ecology, physiology, genetics, applied importance and control. Pensoft Publishers, Sofia. pp. 46-115.

Yocum, G.D. 2001. Differential expression of two HSP70 transcripts in response to cold shock, thermoperiod, and adult diapause in the Colorado potato beetle. Journal of Insect Physiology 47:1139-1145.

Yocum, G.D., Joplin, K.H., and Denlinger, D.L. 1998. Upregulation of a 23 kDa small heat shock protein transcript during pupal diapause in the flesh fly, Sarcophaga crassipalpis. Insect Biochemistry and Molecular Biology 28:677-682.

CHAPTER 2

Diapause-specific gene expression in adults of the northern house mosquito, Culex pipiens L., identified by suppressive subtractive hybridization.

ABSTRACT

In the autumn, males of Cx. pipiens die and the inseminated females enter an overwintering reproductive diapause. In this study, we probe the molecular events underpinning diapause observed in these females. Using suppressive subtractive hybridization (SSH) we have identified 40 genes that are either upregulated or downregulated during this seasonal period of dormancy. These genes can be categorized into eight functional groups: genes with regulatory functions, metabolically-related genes, those involved in food utilization, stress response genes, cytoskeletal genes, ribosomal genes, transposable elements, and genes with unknown functions. Northern blot hybridizations have confirmed the expression of 32 of our SSH clones, including 6 genes that are upregulated specifically in early diapause, 17 that are upregulated in late diapause, and 2 upregulated throughout diapause. In addition, 2 genes are diapause down-regulated and 5 remain unchanged during diapause.

16 INTRODUCTION

One of the primary avian vectors of West Nile virus in the northern United States,

Culex pipiens (L.), enters an adult diapause in late summer and early fall in response to

short daylength and low temperature (Eldridge, 1966; Sanburg and Larsen, 1973;

Spielman and Wong, 1973). The mosquitoes first appear in overwintering sites such as

caves, culverts, and unheated basements (Vinogradova, 2000) as early as August

(Service, 1968; Spielman and Wong, 1973; Onyeka and Boreham, 1987) and remain there until spring when environmental conditions again become favorable for development. Only females enter diapause and most are inseminated prior to entering the hibernation site (Onyeka and Boreham, 1987).

In preparation for diapause, females increase their lipid reserves by feeding on sugar-rich sources such as nectar and rotting fruit (Mitchell and Briegel, 1989a; Bowen,

1992). Although females programmed for diapause can be enticed to take a blood meal by being placed in close proximity to a host (Mitchell, 1983; Mitchell and Briegel,

1989b), it appears this rarely, if ever, happens in the field. Failure of diapausing females to take a blood meal is presumably the reason that so few of the overwintering females harbor West Nile virus (Nasci et al., 2000).

Many aspects of diapause in Cx. pipiens have been well documented. There is a good database that describes the physiological features of this diapause, its environmental regulators, and the hormonal control mechanism. What is currently lacking is an understanding of its molecular underpinning. Very little is known of these events in any

mosquito vector, although such work has been initiated on the embryonic diapause of Oc.

17 triseriatus, an important vector of La Crosse encephalitis (Blitvich et al., 2002). In this

species, several cDNA fragments have been identified using primers designed to amplify

sequences that the La Crosse virus cap-scavenges. Among cDNA fragments that have

been identified are a mitochondrial cytochrome c oxidase subunit, 18S and 28S ribosomal

RNAs, protein disulfide-isomerase, and several novel transcripts, but it is not yet known if any of these genes are indeed involved in regulation of this diapause or in the regulation of viral transcription.

The molecular events involved in the pupal diapause of the flesh fly, are probably the best understood; while many genes are shut down during diapause, a small cluster of genes (approximately 4 %) are diapause upregulated (Denlinger, 2002). Several classes of diapause upregulated genes have been noted, including stress response genes,

developmental arrest genes, and genes involved in regulating specific physiological

activities that are unique to diapause. Although some genes are turned on at the onset of

diapause and remain upregulated until diapause has been broken, others are uniquely

expressed only in early or late diapause. For example, heat shock protein 70 is

upregulated throughout pupal diapause in the flesh fly, Sarcophaga crassipalpis

(Rinehart et al., 2000), while cystatin is upregulated only in early diapause (Goto and

Denlinger, 2002), and ultraspiricle is upregulated only in late diapause (Rinehart et al.,

2001). Other genes, such as the cell cycle regulator proliferating cell nuclear antigen,

are shut down during diapause (Tammariello and Denlinger, 1998).

In this study, suppressive subtractive hybridization (SSH) is used to identify genes

that are differentially expressed during the adult diapause of Cx. pipiens. Expression

18 patterns are confirmed by northern blot hybridization, and the regulated genes that have

been identified are discussed in the context of their possible functional contributions to

diapause.

MATERIALS AND METHODS

Insect Rearing

An anautogenous colony of Cx. pipiens L. was established in September, 2000,

from larvae collected in Columbus, Ohio (Buckeye strain). The colony was maintained

at 25°C, 75% r.h., with a 15hL:9hD daily light:dark cycle. Eggs and first instar larvae were kept under colony conditions until the second instar, and at that time larvae were moved to an environmental chamber at 18°C, 75% r.h., and 15L:9D (nondiapause, 18°C) or placed in an environmental room under diapause-inducing conditions of 18°C, 75% r.h., with a 9L:15D daily light:dark cycle (diapause, 18°C).

Larvae were reared in 5 x 18 x 28 cm plastic containers in de-chlorinated tap water, fed a diet of ground fish food (TetraMin), and maintained at a density of ~250 mosquitoes per pan. Adults were kept in 30.5 cm3 screened cages and provided constant

access to water and honey-soaked sponges. Honey sponges were removed from short-

day cages 10-13 days after adult eclosion to mimic the absence of sugar in the natural

environment during the overwintering period. None of the mosquitoes used in these

experiments were offered a blood meal. To confirm diapause status, primary follicle and

19 germarium lengths were measured, and the stage of ovarian development was determined

according to the methods described by Christophers (1911) and Spielman and Wong

(1973).

Suppressive Subtractive Hybridization

Total RNA was isolated from pools of 20 females by grinding with 4.5 mm

copper-coated spherical balls (“BB’s”) in 1 ml TRIzol® Reagent (Invitrogen). After homogenization, samples were spun at 12,404 g at 4°C for 10 min, and the supernatant was used for RNA extraction following standard protocol (Chomczynski and Sacchi,

1987). RNA pellets were stored in absolute ethanol at -70°C and dissolved in 30 µl ultraPURE™ water (GIBCO) for use in cDNA synthesis. Two rounds of suppressive subtractive hybridization (SSH) were performed using the Clontech PCR-Select™ cDNA

Subtraction Kit to select for genes upregulated in early and late diapause: the first round of SSH consisted of cDNA collected from females in early diapause (short daylength,

18°C, 7-10 days post adult eclosion; tester 1) and early nondiapause (long daylength,

18°C, 7-10 days after adult eclosion; driver 1); the second round of SSH was done using

cDNA from late-diapausing females (short daylength; 18°C, 56-59 days post-adult

eclosion) as tester 2 and early nondiapausing females (long daylength, 18°C, 7-10 days

after adult eclosion) as driver 2. Ovarian dissections of the late-diapausing females (56-

59 days after adult eclosion) indicated that the females were in a late stage of diapause

(Christophers, 1911), just prior to diapause termination.

20 During our initial round of SSH, mRNA was isolated with streptavidin-coupled

paramagnetic particles using the PolyATtract® mRNA Isolation System (Promega), and

this was directly followed by cDNA synthesis according to standard SSH protocol

(Clontech). This yielded a high percentage of clones with identity to fragments of the

16S large ribosomal subunit. To reduce the abundance of 16S during our second round of SSH, mRNA isolation and cDNA synthesis were performed using the BD SMART™

PCR cDNA Synthesis Kit (BD Biosciences) following standard protocol.

Forward and reverse subtracted libraries were cloned using the TOPO TA

Cloning™ Kit (Invitrogen). Transformed plasmids were inserted into competent

Escherichia coli cells and grown overnight on Luria-Bertani (LB) plates containing X-

Gal and ampicillin. For each library, over 100 white colonies were isolated and grown overnight in LB-ampicillin broth at 37°C. Colonies were then purified with QIAprep

Spin Miniprep (QIAGEN), run on a 1% agarose gel to determine concentration, and sequenced using the vector internal primer sites (T7 and M13R) at the Ohio State

University Plant-Microbe Genomics Facility on an Applied Biosystems 3730 DNA

Analyzer using BigDye® Terminator Cycle Sequencing chemistry (Applied Biosystems) following manufacturer’s protocol.

Northern Blot Analysis

Fifteen micrograms of denatured total RNA samples were separated by electrophoresis on a 1.4% agarose denaturing gel (0.41 M formaldehyde, 1X MOPS-

EDTA-sodium acetate). Visualization of ethidium bromide stained rRNA under UV light

21 exposure was used to confirm equal loading. Following the TURBOBLOTTER™ Rapid

Downward Transfer Systems protocol (Schleicher and Schuell), the RNA was transferred for 1.5 hours onto a 0.45 micron MagnaCharge nylon membrane (GE Osmonics) using downward capillary action in 3 M NaCl, 8 mM NaOH transfer buffer, followed by neutralization in a 1 M phosphate buffer solution and UV crosslinking. The membrane was then air-dried and either stored at -20°C or used immediately for hybridization.

Digoxigenin (DIG)-labeled cDNA probes were developed from genes of interest in our forward and reverse subtracted SSH libraries. PCR was performed on each clone using the SSH nested primers (Clontech PCR-Select™ cDNA Subtraction Kit) according to the following parameters: 94°C for 3 min and 35 cycles of 94°C for 30 sec, 60°C for

30 sec, and 72°C for 2 min, followed by a 7 min extension at 72°C and a 4°C hold. The

PCR products were run on a 1% TAE agarose gel and the band of interest was excised from any remaining vector, extracted with Ultrafree®-DA (Millipore), and re-amplified by PCR. To confirm clone identity, PCR products were sequenced using the forward and reverse nested primers (Clontech) by the methods described above. The cDNAs were individually labeled in an overnight DIG reaction using 100ng of template DNA and the

Dig High Prime DNA Labeling and Detection Starter Kit II (Roche Applied Sciences).

Probes were stored at -20°C.

Hybridization was carried out overnight followed by stringency washes and immunological detection using the Dig High Prime DNA Labeling and Detection Starter

Kit II (Roche Applied Sciences) according to manufacturer’s protocol. Blots were then exposed to chemiluminescence film (Kodak Biomax). Each northern was replicated three

22 or more times. To confirm equal transfer of RNA, each membrane was stripped with 0.2

M NaOH/0.1% SDS and re-probed using DIG-labeled 28S cDNA, according to

manufacturer’s instructions.

Bioinfomatics Analyses

Sequences were edited and assembled using dnaLIMS (dnaTools) and the Baylor

College of Medicine Search Launcher: Sequence Utilities

(http://dot.imgen.bcm.tmc.edu/seq-util/seq-util.html). Similar sequences and percent identities were determined by BLASTn and BLASTx searchs in GenBank

(http://www.ncbi.nlm.nih.gov/). Low percent identities (<40%) and short matches (<30 bp) were considered not significantly similar and are thus listed as “genes with unknown function”. Only BLASTn results were listed if this search produced a high percent identity, otherwise BLASTx results were used. Both BLASTn and BLASTx identifications were listed in Tables 1 and 2 if this provided additional useful information.

Nucleotide sequences were deposited in GenBank and assigned accession numbers listed in Tables 1 and 2.

RESULTS

Early and Late Diapause Subtraction

Our first round of SSH (early diapause – early nondiapause), yielded only 5 unique diapause-upregulated clones from our initial screening of 48 clones, while the rest were identified as fragments of 16S large ribosomal subunit (with high identity to Aedes

23 aegypti large ribosomal subunit RNA gene, AY431935). The 16S sequences were all of similar length, corresponding to two major bands on the final SSH PCR gel. Therefore, before continuing with sequencing of additional clones, we constructed new libraries from selected regions of this gel, excluding the putative 16S large ribosomal subunit fragment bands. Sequencing of 95 randomly chosen clones from each forward and reverse subtracted library was much more successful, resulting in 18 unique clones that were further analyzed by northern blot hybridization.

Our second round of SSH utilized cDNA from Cx. pipiens in late-diapause (56-

59 days post adult eclosion) as the tester and cDNA from nondiapausing females (7-10 days after adult eclosion) as the driver. When constructing the late-diapause subtracted libraries, a slightly different method was employed. Rather than rely on mRNA purification to eliminate ribosomal RNA, the SMART cDNA synthesis kit (BD

Biosciences) was used to create full-length enriched cDNA pools, which were then used in the subsequent subtraction procedures. While 10 of these clones had sequences with high similarity to the 16S large ribosomal subunit gene, the remaining clones appeared to be of mRNA origin. Out of 95 clones sequenced (80 from the forward-subtracted library and 15 from the reverse-subtracted library), 22 were unique clones, and all but one were detectable by northern blot hybridization.

Confirmation by Northern Blot Analysis

Northern blot hybridizations were used to confirm the putative upregulation or downregulation of 40 cDNAs obtained in our early- and late-diapause subtracted libraries

24 (Figures 2.1 and 2.2). In most cases, northern blot hybridization confirmed the upregulation or downregulation of the cDNAs that were isolated, however, in a few cases northern blots showed a different pattern of expression than that obtained by SSH. All clones produced bands of expected size, as determined by information retrieved from

GenBank. Our early-diapause clones ranged in size from 177 to 408 bp while our late- diapause clones ranged from 133 to 1,450 bp. The highest matching sequences retrieved from GenBank, including percent identities, organism, and accession numbers, are listed in Tables 2.1 and 2.2. Several clones isolated by SSH were undetectable by northern blots, suggesting that a more sensitive technique such as real time PCR will be needed to confirm the diapause expression pattern suggested by SSH.

Diapause Upregulated Genes

Our SSH library results for early diapause yielded 18 unique clones, 11 of which were verifiable by northern blot hybridization (Figure 2.1). The following genes appear to be upregulated only in early diapause: 2 genes with regulatory functions (CpiED-A38, cytosolic small ribosomal subunit S3A; CpiED-L01, ribosomal protein S6), one gene

involved in food utilization (CpiED-A47, fatty acid synthase), one with a metabolic

function (CpiED-A07, L-malate dehydrogenase), a cytoskeletal gene (CpiED-A09,

muscle-specific actin 2), and one ribosomal gene (CpiED-A32, large ribosomal subunit

L18). Three genes encoding ribosomal protein S24 (CpiMD-C04), cytochrome oxidase subunit I (CpiMD-H06), and large ribosomal subunit (CpiMD-A01) were obtained from

our late-diapause library, but northern blots indicated that they are only upregulated in

25 early diapause. In addition, 7 genes are putatively upregulated according to SSH, but

these genes were expressed at levels undetectable by northern blot hybridization. These

include transcription elongation factor polypeptide B (CpiED-B03), thyroid hormone

receptor-associated protein TRAP170 (CpiED-D03), Methoprene-tolerant protein

(CpiED-M01), selenoprotein (CpiED-A24), disease resistance Cf-2 protein (CpiED-

C09), small ribosomal subunit 27A (CpiED-E08), and a gene with unknown function

(CpiED-A01).

We have also obtained 22 clones from our late-diapause upregulated library

(Figure 2.1), all of which were verifiable by northern blot hybridization, except for one clone, calreticulin (CpiMD-G10). Although some clones were expressed in both early and late diapause, others were upregulated only in late diapause. Genes upregulated only in late-diapause include two stress response genes (CpiMD-A06, aldehyde oxidase and

CpiMD-A11, heat shock protein 23), a gene with metabolic function (CpiMD-E09, methylmalonate-semialdehyde dehyrogenase), a ribosomal gene from the endosymbiont

Wolbachia pipientis (CpiMD-C02, 23S ribosomal RNA gene), two transposable elements

(CpiMD-D02, trasposon T1-2; CpiMD-D11, transposable element Mimo-Cp2), and 8 genes with unknown functions (CpiMD-B02, -B11, -D06, -F01, -F03, -F07, -and –H02).

In addition, 2 genes were obtained from our late-diapause library but are upregulated in both early and late-diapause. These genes encode cytochrome oxidase subunit 3

(CpiMD-H04) and a gene with unknown function (CpiMD-M43).

26 Diapause Downregulated Genes and Genes Unchanged in Diapause

Only two genes of interest were obtained from our reverse subtracted library as

being downregulated in early diapause (Figure 2.2): CpiED-A15 encoding trypsin and

CpiED-A34 encoding serine protease. These results were confirmed by northern blot

hybridization; both yielded a strong signal in nondiapausing females, no signal in early

diapause, and only a weak signal in late diapause.

In addition, several obtained from our forward and reverse subtracted libraries

showed no change in expression levels in all three stages tested (Figure 2.2). These include genes encoding poly A binding protein (CpiED-A18), ubiquitin extension protein

(CpiED-A29), cecropin A (CpiMD-A10), isoform C beta tubulin 56D (CpiED-B06), and

28S large subunit ribosomal RNA gene (CpiMD-H09). The consistency of 28S

expression prompted us to use this gene as a control for the northern blot hybridizations.

DISCUSSION

The results presented here provide some first clues about the molecular events that characterize the adult diapause of Cx. pipiens. Using SSH, we have identified 40 genes differentially expressed in diapause, and confirmed the following expression patterns by northern blot hybridization: 6 genes are upregulated specifically in early diapause, 17 genes are upregulated in late diapause, and 2 genes are upregulated throughout diapause. In addition, we have identified 2 genes that are diapause downregulated and 5 that remained unchanged during diapause. We have categorized these genes into 8 distinct groupings: regulatory function, food utilization, stress response,

27 metabolic function, cytoskeletal, ribosomal, transposable elements, and genes with unknown functions. Northern blot hybridization has confirmed the expression of 32 of the 40 genes obtained by SSH, while the others appear to be expressed at levels undetectable by this method.

Regulatory Genes

The diapause of Cx. pipiens is characterized by a state of inactivity and a dramatically slow rate of ovarian maturation (Readio et al., 1999). Genes regulating these and other molecular events may prove useful in understanding how Cx. pipiens can survive in a prolonged inactive state. Certain ribosomal proteins have functions in regulating cell growth and death in addition to their roles in translation (Naora and Naora,

1999). The appearance of three such ribosomal proteins in our SSH libraries suggests a possible contribution of these proteins in regulating the adult diapause of Cx. pipiens.

Ribosomal protein (rp) S3A, rpS6, and rpS24 are all components of the 40S ribosomal subunit mRNA binding domain and are involved in the initiation of translation

(Takahashi et al., 2002). Two of these ribosomal proteins are expressed at low levels in early diapause and all three become highly expressed in late diapause, shortly before diapause is terminated.

The highly conserved gene encoding rpS3A is found in high concentration in the ovaries of Anopheles gambiae (Zurita et al., 1997) and in the follicular epithelial cells of

Drosophila melanogaster (Reynaud et al., 1997). Suppression of rpS3A in D. melanogaster leads to a disruption of the follicular epithelium and an inhibition of ovarian development (Reynaud et al., 1997). Since ovarian development requires high

28 protein synthesis, suppression of this gene likely leads to a disruption in this process.

Since arrested ovarian development is a prominent characteristic of Cx. pipiens diapause

(Spielman and Wong, 1973), the lack of expression of rpS3A in early diapause may be key to this developmental arrest.

Of equal interest is the undetectable level of expression of ribosomal protein S6

(rpS6) in early diapause and its subsequent upregulation in late diapause. This is another

gene that is upregulated prior to oogenesis in Ae. aegypti; rpS6 mRNA accumulates 24-

48 h after adult eclosion, remains stable until the blood meal prompts protein synthesis in

the fat body (Niu and Fallon, 2000). Of particular interest to the diapause of Cx. pipiens

is the fact that suppression of rpS6 activity has been implicated in other models of

developmental arrest. In the encysted embryos of the brine shrimp Artemia franciscana,

S6 kinase, which is required for S6 phospohrylation, shows a rapid accumulation of

mRNA 4 h after embryos are placed in hatching conditions, and the active enzyme is

detectable within 15 minutes of diapause break (Santiago and Sturgill, 2001). While

embryonic diapause differs greatly from an adult diapause, the two models suggest that

downregulation of rpS6 may be a key element in arrested development, and its

upregulation may be essential for diapause termination.

The upregulation of our clone with high similarity to the Methoprene tolerant

gene (Met) from Drosophila is of particular interest because it is possibly a juvenile

hormone receptor (Wilson, 2003) and may function as a JH-dependent transcription

factor (Miura et al., 2005). The diapause of Cx. pipiens is known to be regulated by an

absence of juvenile hormone (Readio et al., 1999), thus the upregulation of Met during

29 diapause is puzzling because one might have anticipated that, if anything, Met would be downregulated at this time. The link between Met expression and the juvenile hormone mediation of diapause is unclear at this point, but it raises intriguing scenarios that may need to be considered for future work on the hormonal control of diapause in this species.

In addition, several other genes obtained by SSH may also have regulatory functions. A gene encoding calreticulin, a multi-functional Ca2+ binding protein, was obtained from our late-diapause library. Calreticulin acts as a molecular chaperone in

Ca2+-dependent pathways through calcium binding (Michalak et al., 2002), functions in the immune system (Henson et al., 2001; Johnson et al., 2001; Gao et al., 2002), and can regulate gene expression (Michalak et al., 1999). Two other putative diapause- upregulated genes encoding thyroid hormone receptor-associated protein (TRAP 170) and predicted transcription elongation factor polypeptide B are likely involved in signal transduction (Malik and Roeder, 2000) and mRNA processing (Shilatifard, 2004), respectively.

Food Utilization

A key characteristic of diapausing Cx. pipiens is that they lack the host-seeking response and will not take a blood meal under natural conditions (Mitchell, 1983; Bowen et al., 1988). Although diapausing females can be enticed to take a blood meal if the host-seeking step is bypassed (Mitchell, 1983), most of the blood ingested is ejected within 24 h and blood that remains in the gut is used neither for sequestration of lipid reserves nor for vitellogenesis (Mitchell and Briegel, 1989b). Instead, females are

30 programmed to sequester lipid reserves (Bowen, 1992; Mitchell and Briegel, 1989a) and

do so by feeding on plant sources rich in carbohydrates such as nectar and rotting fruits.

Our results show the downregulation of two genes encoding the blood digestive enzymes

trypsin and serine protease in early diapause, while the gene encoding the enzyme

involved in the conversion of sugars to lipid stores, fatty acid synthase, is highly

upregulated at this time. The evidence presented here supports the contention that even if

a blood meal is taken by diapausing Cx. pipiens, they lack the molecular machinery

required for blood digestion and are instead programmed to sequester lipid reserves. In late diapause, the accumulation of mRNAs encoding trypsin and serine protease may indicate that females are preparing to terminate diapause by regaining competence to digest a blood meal.

Stress Response

Overwintering insects confront harsh environmental conditions including low temperature, varying relative humidity, and invasion by pathogenic organisms. In several insect species, heat shock proteins are highly upregulated upon entry into diapause

(Denlinger et al., 2001). These proteins act as molecular chaperones by preventing abnormal protein folding during environmental stresses such as extreme heat, cold, or desiccation and have also been implicated in playing a role in cell cycle arrest (Feder et al., 1992). In the pupal diapause of the flesh fly Sarcophaga crassipalpis, hsp23 and

hsp70 are developmentally upregulated upon the entry into diapause and remain

expressed until diapause has been broken (Rinehart et al., 2000; Yocum et al., 1998),

31 while hsp90 is downregulated at this time but remains responsive to environmental stress

(Rinehart and Denlinger, 2000). In Cx. pipiens, hsp23 is upregulated in late diapause, but the upregulation is slight by comparison with the strong upregulation of hsp23 noted in S. crassipalpis (Yocum et al., 1998). A slight elevation of hsp70 was also noted in the adult diapause of the Colorado potato beetle (Yocum, 2001), suggesting that upregulation of hsps is not a major component of the diapause syndrome in adults. In adults of

Drosophila triauraria, hsps do not appear to be at all upregulated during diapause (Goto and Kimura, 2004)

A second stress response gene identified in late diapause is aldehyde oxidase, which encodes a multifunctional molybdo-flavoenzyme with broad substrate specificity involved in the oxidation of aromatic N-heterocycles and aldehydes (Garattini et al.,

2003). Several functions have been proposed for this enzyme including its involvement in catalyzing metabolic pathways, vitamin degradation, and detoxification of environmental pollutants (Gerattini et al., 2003). In addition, aldehyde oxidase may play an important role in insecticide resistance in the common house mosquito, Cx. quinquefasciatus (Coleman et al., 2002); our clone from Cx. pipiens shares 93% identity with aldehyde oxidase from Cx. quinquefasciatus. In certain insecticide-resistant strains of Cx. quinquefasciatus, the aldehyde oxidase gene is amplified in conjunction with two resistance-associated esterases, and the enzyme shows high substrate specificity for insecticide oxidation (Hemingway et al., 2000). Thus, aldehyde oxidase may also function in diapausing Cx. pipiens to oxidize environmental pollutants found in overwintering sites.

32 Two additional stress-response genes, selenoprotein and a disease resistance Cf-2 protein, are putatively upregulated in Cx. pipiens diapause and may confer protection against environmental stress. In D. melanogaster, selenoproteins function as antioxidants and can decrease lipid peroxidation (Morozova et al., 2003), functions that may be especially important in long-lived individuals that are in diapause. In Arabidopsis thaliana, the Cf-2-dependent disease resistance protein is involved in a fungal defense pathway (Kruger, et al., 2002). Field studies with diapausing Cx. pipiens indicate that two fungi, Cephalosporium sp. and Entomophthera sp., cause considerable mortality in overwintering populations from England (Service, 1968), thus suggesting the importance of this gene in overwintering mosquito populations.

Although cecropin A and ubiquitin extension protein were obtained from our late- diapause upregulated library, northern blot hybridizations indicate that their expression levels remain unchanged in diapause. The immune peptide, cecropin A, was detectable at very low levels in all stages tested. Further studies are needed to demonstrate if it is upregulated in response to a bacterial infection in overwintering females. The low level of expression is consistent with that observed in other species: Bartholomay et al. (2003) demonstrated that cecropin A transcripts are not detectable in naïve mosquitoes, but are rapidly transcribed after bacterial inoculation. Likewise, in spite of its isolation from our diapause library, ubiquitin extension protein, a gene associated with protein degradation and stress responses (Esser et al., 2004), was expressed equally in nondiapausing and diapausing mosquitoes, as noted with northern blots.

33 Metabolic Genes

Four genes with metabolic functions are upregulated during diapause: L-malate

dehydrogenase, methylmalonate-semialdehyde dehydrogenase, cytochrome oxidase (CO)

subunit III and COI. Two of these, COI and COIII, are of mitochondrial origin and serve

an essential role in aerobic oxidation. Although metabolic rates in insects are typically

suppressed during diapause, the metabolic suppression in adult diapauses is not as

extensive as in other stages such as the egg or pupa (Danks, 1987). The upregulation of

the two mitochondrial genes, COI and COIII, in early Cx. pipiens diapause may, at first,

seem counterintuitive, but Cx. pipiens adults are quite active prior to hibernation. They

actively seek sugar meals (Bowen, 1992), and they also must fly to their hibernation site.

Diapause preparation thus requires considerable energy. Similar results have been noted

in the early phase of larval diapause in the Japanese beetle, Popillia japonica, where

cytochrome oxidase activity actually increases during early diapause (Ludwig, 1953). In

Cx. pipiens, COI expression is depressed in late diapause, while COIII transcripts remain high.

The concurrent upregulation of L-malate dehydrogenase (MDH) and

methylmalonate semialdehyde dehydrogenase may also be involved in specific metabolic

events associated with diapause. MDH has been implicated in increased cold tolerance;

certain forms of this enzyme function more efficiently at low temperatures (Kim et al.,

1999). MDH upregulation in conjunction with increased cold tolerance has been

observed in organisms as diverse as the channel catfish Ictalurus punctatus (Seddon and

34 Prosser, 1997) and the potato Solanum sogarandinum (Rorat et al., 1997). It is not clear

what unique function methylmalonate semialdehyde dehydrogenase, an enzyme involved

in amino acid metabolism, may play during diapause.

Cytoskeletal Genes

Our experiments indicate that the expression of cytoskeletal genes is affected by

diapause: an actin is upregulated in early diapause and returns to low levels by late

diapause, while a beta tubulin is unchanged during diapause. The upregulation of an

actin in early diapause is in contrast to reports from other species, but this may reflect the

increased flight activity of females preparing for hibernation. A brain-specific actin is

downregulated during the pharate larval diapause of the gypsy moth Lymantria dispar

(Lee et al., 1998). Data from plant models indicate that actin downregulation may

contribute to the increased cold tolerance associated with dormancy. In wheat (Triticum aestivum), actin depolymerizing factor (ADF) accumulation is a major component of cold

acclimation (Ouellet et al., 2001). Upon activation, this protein sequesters actin and

induces actin depolymerization (Ouellet et al., 2001), and removal of actin from the

cytoskeleton increases membrane fluidity and thus increases resistance to cold. In Cx.

pipiens, however, not only do females actively fly during diapause preparation, but they

continue to move around within their hibernaculum during the winter months (Minar and

Ryba, 1971; Buffington, 1972).

35 Ribosomal Genes

In addition to the three ribosomal genes thought to serve regulatory functions (see

“Regulatory Genes”), three other ribosomal genes are upregulated in early diapause:

large ribosomal subunit L18, small ribosomal subunit 27A, and large ribosomal subunit

(16S). The fact that two of these ribosomal genes are downregulated in late diapause

(L18 was undetectable by northern blots) suggests that their function is restricted to the events of early diapause. By contrast, the 23S ribosomal RNA gene was recovered in late

diapause from the obligate intracellular bacteria of Cx. pipiens, Wolbachia. The strong upregulation of this gene in late diapause indicates that Wolbachia is active in late- diapausing Cx. pipiens. The differential expression of this Wolbachia gene in association with the diapause of its host suggests that the diapause status of Cx. pipiens may affect development of this bacterial parasite, as demonstrated in Wolbachia-infected eggs during the diapause of another mosquito, Ae. albopictus (Ruang-areerate et al., 2004).

Transposable Elements

Curiously, two genes encoding fractions of transposable elements, transposon

T1-2 and Mimo-Cp2, are upregulated during late diapause in Cx. pipiens. Although the function of transposable elements in diapause is unclear, diapause regulation of transposons has also been noted in two other species: two genes encoding retroviral envelope proteins are expressed during the embryonic diapause of Bombyx mori

(Yamashita et al., 2001), and a gene encoding a retrotransposon is highly expressed in the

36 early pupal diapause of S. crassipalpis (Denlinger, 2002). That transposable elements

would be diapause upregulated in all three of these species suggests an intriguing, but

still unknown, role for transposable elements in the regulation of diapause.

Genes with Unknown Function

Nine genes with unknown functions are upregulated in late diapause, as confirmed by northern blots, and one of these (CpiMD-M43) is also expressed in early diapause. This gene is of particular interest since its high level of expression in early

diapause suggests it may play a role in initiating the diapause program.

In summary, this study represents the first large-scale investigation of the

molecular aspects of diapause in any mosquito species. By suppressive subtractive

hybridization, we have demonstrated the differential regulation of genes specifically involved in early and late diapause and have categorized these genes into several distinct functional groups. Future work will certainly reveal additional genes and possibly additional gene categories that are involved in the diapause of Cx. pipiens. We anticipate that these results will be useful in probing potential molecular commonality in the diapauses of different taxa and developmental stages. We also anticipate that this type of work will prove helpful in understanding the transseasonal maintenance of West Nile virus in diapausing Cx. pipiens and may contribute to an understanding of the dynamic relationships between other pathogens and their vectors during the overwintering season.

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X X X X X X X X X X X XX X X X northern northern

gov/BLAST/). In the blast , AF202953 , AAG02462 , T13418 , AE017256

, AAC63387 r r r , AY441061 , AAM14384.1 , U23710 , AF457547 , AF417833 , AF417833 ractive hybridization. Percent ractive hybridization. Percent

, AY432783 , XP_143238.2 , AY431319 , AY431545 , AY431935 , AY431732 , AY431617 , AAX55681 , AY064700 , AY289764 , AY433139 , AF466603 , AF425847 (18°C, short daylength; 7-10 days post LASTn) and/or translation (BLASTx) LASTn) and/or translation (BLASTx)

diapause, ED and LD, respectively. edes aegypti us musculus us Armigeres subalbatus Drosophila melanogaste Drosophila Anopheles gambiae Arabidopsis thaliana Arabidopsis Culex pipiens quinquefasciatus Culex pipiens Aedes aegypti Aedes Aedes aegypti Aedes Aedes aegypti Aedes melanogaste Drosophila M Wolbachia pipientis Wolbachia Aedes aegypti Aedes aegypti Aedes Culex tarsalis Uranotaenia lowii Uranotaenia Sarcophaga crassipalpis Sarcophaga Aedes aegypti Aedes Aedes aegypti Aedes Aedes aegypti Aedes Aedes aegypti Aedes melanogaste Drosophila Aedes aegypti Aedes

identity, organism, accession # accession identity, organism, ED LD 85%, 59%, 87%, 42%, 93%, 91%, 86%, 100%, A 98%, 97%, 97%, 97%, 87%, 92%, 87%, 49%, 40%, 77%, 98%, 89%, 92%, 85%, 94%,

Table 1.1 (continued). (continued). 1.1 Table

GenBank (http://www.ncbi.nlm.nih. through a nucleotide (B , isolated by suppressive subt

Cx. pipiens n blot is indicated by an “X” in early

ylength; 56-59 days post adult eclosion)

sion #’s were retrieved from 23S ribosomal RNA generibosomal 23S n

l tress response tress ood utilization CpiED-A47 230 synthase fatty-acid n CpiED-D03 614TRAP170 protein receptor-associated hormone thyroid x CpiED-A24 306 selenoprotein n CpiED-C09 306 Cf-2 protein resistance disease putative x CpiMD-A06 1,450 oxidase aldehyde n regulatory CpiED-A38 469 S3A subunit ribosomal small cytosolic n Diapause Upregulated Genes clone size (bp) putative identityf s blast % CpiED-A32 177 L18 subunit ribosomal large cytosolic n CpiED-E08CpiMD-A01CpiMD-C02 105 231 324 27A subunit ribosomal small cytosolic RNA subunit ribosomal large endosymbiont Wolbachia x n n CpiED-L01 296 S6 protein ribosomal CpiMD-H04 805 3 subunit oxidase cytochrome CpiMD-H06ribosoma 912 I subunit oxidase cytochrome n CpiMD-H09 291 RNA gene ribosomal subunit large 28S n CpiMD-A11 483 protein shock 23kDa heat x CpiED-B03 356 B polypeptide factor elongation transcription predicted x CpiED-M01 70 protein methoprene-tolerant putative x CpiMD-C04 295 S24 protein ribosomal n cytoskeletal CpiED-A09 408 2 actin muscle-specific n metabolic function metabolic CpiED-A07CpiMD-E09 199 1,055 dehydrogenase L-malate dehydrogenase putative methylmalonate-semialdehyde x n CpiMD-G10 519 calreticulin putative

adult eclosion) and late (18°C, short da identities, organisms, and acces column, “n” and “x” indicate putative identities obtained query, respectively. Confirmation by norther Table 2.1. Diapause upregulated genes from 44

X X X X X X X X X X X

XX northern

, B34751 , EAA13087 y y y y y y y y y , AF217612

Anopheles gambiae Anopheles Culex pipiens gambiae Anopheles 44%, 87%, 65%, n,xn,x significant no similarit n,x significant no similarit n,x significant no similarit n,x significant no similarit significant no similarit n,xn,x significant no similarit n,x significant no similarit n,x significant no similarit significant no similarit

y y y y y y y y y

Diapause Upregulated Genes Continued Genes Upregulated Diapause clone elements transposable CpiMD-D02 (bp) size identity putative 897 T1-2 transposon blast % # accession identity,organism, x ED LD CpiMD-D11 function unknown with genes CpiED-A01 889 Mimo-Cp2 element transposable 859 similarit significant no n CpiMD-B02CpiMD-B11CpiMD-D06CpiMD-F01CpiMD-F03 312CpiMD-F07 841 1,047CpiMD-H02 similarit significant no CpiMD-H03 445 similarit significant no similarit CpiMD-A43 significant no 311 250 similarit significant no 312PEST str. gambiae Anopheles 133 similarit significant no 561 similarit significant no similarit significant no similarit significant no x Table 2.1 (continued).

Diapause Downregulated and Genes Unchanged in Diapause northern clone size (bp) putative identity blast % identity, organism, accession # ND ED LD

Diapause Downregulated Genes food utilization CpiED-A15 294 serine protease x 40%, Anopheles gambiae , AAA73920 X CpiED-A34 264 trypsin n 98%, Culex pipiens pallens , AY034060 X

Genes Unchanged in Diapause regulatory CpiED-A18 292 putative poly A binding n 91%, Aedes aegypti , AY431644 XXX stress response CpiED-A29 295 ubiquitin extension protein n 94%, Drosophila melanogaster , X59943 XXX CpiMD-A10 333 cecropin A n 79%, Culex pipiens pipiens , AY189808 XXX cytoskeletal CpiED-B06 241 isoform C (betaTub56D) n 89%, Drosophila melanogaster , NM_166357 XXX

Table 2.2. Diapause downregulated genes from Cx. pipiens, isolated by suppressive subtractive hybridization, and genes unchanged in diapause. Percent identities, organisms, and accession #’s were retrieved from GeneBank (http://www.ncbi.nlm.nih.gov/BLAST/). In the blast column, “n” and “x” indicate putative identities obtained through a nucleotide (BLASTn) and/or translation (BLASTx) query, respectively. Confirmation by northern blot is indicated by an “X” in nondiapause (18°C, long daylength; 7-10 days post adult eclosion) and early diapause (18°C, short daylength; 7-10 days post adult eclosion), and late diapause (18°C, short daylength; 56- 59 days post adult eclosion).

short Mimo-Cp2 28S CpiMD-D11 on). Each on). Each

CpiMD-M43

LD ED ND CpiMD-D02 transposon T1-2 LD ED ND LD ED ND G. Transposable Elements G. Transposable actin CpiMD-H03 ND ED LD ED ND CpiED-A09 ND ED LD ED ND D. Cytoskeletal

CpiMD-C02 23S (Wolbachia) ND ED LD ED ND

are listed above each nd putative identities CpiMD-H02 ND ED LD ED ND hsp23 CpiMD-A11 after adult eclosi days 56-59 t daylength; ND ED LD ED ND CpiMD-A01 ND ED LD ED ND large ribosomal subunit ribosomal large CpiMD-F07

LD ED ND

CpiMD-A06 ng was confirmed by northern blot hybridization with a hybridization northern blot ng was confirmed by ND ED LD ED ND aldehyde oxidase C. StressC. Response CpiED-A32 ys after adult eclosion), ED = females in early diapause (18°C,

ND ED LD ED ND ribosomal subunit L18 subunit ribosomal

CpiMD-F03 LD ED ND

upregulated SSH clones. Clone ID a

CpiED-A47 ND ED LD ED ND fatty acid synthase acid fatty

B. Food Utilization Food B. CpiMD-F01 CpiMD-H06 ND ED LD ED ND LD ED ND cytochrome c oxidase I Cx. pipiens LD = females in late diapause (18°C, shor

CpiMD-C04 CpiMD-H04 CpiMD-D06 ND ED LD ED ND LD ED ND ribosomal protein S24 protein ribosomal cytochrome oxidase c 3

CpiED-L01 CpiMD-B11 CpiMD-E09 ND ED LD ED ND LD ED ND ribosomal protein S6 protein ribosomal meth. dehydrogenase meth. g of total RNA pooled from 20 females. Equal loadi g of total

CpiED-A38 CpiED-A07 CpiMD-B02

ND ED LD ED ND LD ED ND LD ED ND LD ED ND LD ED ND malate dehydrogenase ribosomal subunit S3A subunit ribosomal A. Regulatory FunctionE. Metabolic Function Unknown with Genes H. F. Ribosomal Diapause Upregulated Genes Upregulated Diapause 28S 28S 28S of hybridization Northern blot Figure 2.1. cDNA probe. lane contains 15 blot. ND = nondiapausing females (18°C, long daylength; 7-10 da 7-10 daylength; females (18°C, long blot. ND = nondiapausing days after adult eclosion), 7-10 daylength;

Diapause Downregulated Genes

A. Food Utilization CpiED-A15 CpiED-A34 serine protease trypsin

28S

ND ED LD ND ED LD

Genes Unchanged in Diapause

B. Regulatory C. Stress Response D. Cytoskeletal CpiED-A18 CpiED-A29 CpiMD-A10 CpiED-B06 poly A binding ubiquitin cecropin A beta tubulin

28S

ND ED LD ND ED LD ND ED LD ND ED LD

Figure 2.2. Northern blot hybridization of Cx. pipiens diapause downregulated genes and genes unchanged in diapause. Clone ID and putative identities are listed above each blot. ND = nondiapausing females (18°C, long daylength; 7-10 days after adult eclosion), ED = females in early diapause (18°C, short daylength; 7-10 days after adult eclosion), LD = females in late diapause (18°C, short daylength; 56-59 days after adult eclosion). Each lane contains 15 µg of total RNA pooled from 20 females. A 28S cDNA probe was used to confirm equal loading.

Diapause Downregulated and Genes Unchanged in Diapause northern clone size (bp) putative identity blast % identity, organism, accession # ND ED LD

Diapause Downregulated Genes food utilization CpiED-A15 294 serine protease x 40%, Anopheles gambiae , AAA73920 X CpiED-A34 264 trypsin n 98%, Culex pipiens pallens , AY034060 X

Genes Unchanged in Diapause regulatory CpiED-A18 292 putative poly A binding n 91%, Aedes aegypti , AY431644 XXX

stress response CpiED-A29 295 ubiquitin extension protein n 94%, Drosophila melanogaster , X59943 XXX CpiMD-A10 333 cecropin A n 79%, Culex pipiens pipiens , AY189808 XXX

cytoskeletal CpiED-B06 241 isoform C (betaTub56D) n 89%, Drosophila melanogaster , NM_166357 XXX

Table 2. Diapause downregulated genes from Cx. pipiens, isolated by suppressive subtractive hybridization, and genes unchanged in diapause. Percent identities, organisms, and accession #’s were retrieved from GeneBank (http://www.ncbi.nlm.nih.gov/BLAST/). In the blast column, “n” and “x” indicate putative identities obtained through a nucleotide (BLASTn) and/or translation (BLASTx) query, respectively. Confirmation by northern blot is indicated by an “X” in nondiapause (18°C, long daylength; 7-10 days post adult eclosion) and early diapause (18°C, short daylength; 7-10 days post adult eclosion), and late diapause (18°C, short daylength; 56-59 days post adult eclosion).

CHAPTER 3

Diapause in the mosquito Culex pipiens evokes a metabolic switch that shuts down genes

encoding blood digestive enzymes and upregulates a gene associated with sugar

utilization and lipid storage.

ABSTRACT

A key characteristic of diapause in Culex pipiens is the switch from blood-feeding in nondiapausing individuals to sugar feeding in diapause-destined females. We present evidence demonstrating that genes encoding enzymes needed to digest a blood meal

(trypsin and a chymotrypsin-like protease) are downregulated in prehibernating females, and concurrently a gene associated with the accumulation of lipid reserves (fatty acid synthase) is highly upregulated. As females then enter the hibernation state (diapause) fatty acid synthase is only sporadically expressed, and expression of trypsin and chymotrypsin-like remain undetectable. Late in diapause (2-3 months at 18°C) the genes

encoding the digestive enzymes begin to be expressed as the female prepares to take a

blood meal upon termination of diapause. The results thus underscore a molecular switch

that either directs the female towards blood feeding (nondiapause) or sugar feeding and

lipid sequestration (diapause).

50 INTRODUCTION

The northern house mosquito, Culex pipiens (L.), plays a prominent role in maintaining the natural reservoirs of several arthropod-borne viruses including St. Louis

and West Nile viruses in North America. The ecology and physiology of Cx. pipiens

have therefore been extensively studied, with particular interest being paid to the

overwintering stage. Winter is typically characterized by a significant decrease or

absence of vector-borne diseases, and several studies have shown that the overwintering

stage of the mosquito can harbor pathological agents during this time and thus reinitiate

disease transmission the following spring (Bellamy et al., 1958; Watts et al., 1974;

Reisen et al., 2002). This appears to be a likely scenario explaining the reappearance of

West Nile virus in the New York City area following the initial outbreak in the summer

of 1999 (Nasci et al., 2001). In this case, diapausing adult females of Cx. pipiens were

found to harbor the virus during the winter months, and such mosquitoes may have

reintroduced the virus into bird populations the following season.

Cx. pipiens is a temperate zone species that enters an overwintering dormancy

(diapause) in response to short daylength and low temperatures received in the fourth

larval instar and early pupal stage (Eldridge, 1966; Sandburg and Larsen 1973; Spielman

and Wong 1973). Diapausing individuals first appear in overwintering sites such as

caves, culverts and unheated basements (Vinogradova, 2000) as early as August

(Spielman and Wong, 1973; Onyeka and Boreham, 1987). Only adult females enter

diapause; males die after fertilizing the females in the autumn (Service, 1968; Onyeka

and Boreham, 1987). One of the main features of diapause is that the primary ovarian

51 follicles remain in a state of arrested development (Eldridge, 1966; Sanburg and Larsen,

1973; Spielman and Wong 1973). In addition, a number of behavioral changes occur,

including a lack of host-seeking behavior and the concurrent increase in feeding on

carbohydrate-rich nectar, rotting fruits, and other plant products, which leads to a hypertrophy of the fat body prior to the onset of diapause (Mitchell, 1983; Mitchell and

Briegel, 1989a; Bowen, 1992).

Females programmed for diapause can be enticed to take a blood meal under laboratory conditions if placed in close proximity to a host (Eldridge and Bailey, 1979;

Bailey et al., 1982; Mitchell and Briegel, 1989b), but most of the blood ingested by diapausing females is ejected. Blood that remains in the midgut is not used to increase lipid reserves, and only a few females have been observed to use this blood to initiate vitellogenesis (Mitchell and Briegel, 1989b). In diapause, females of Cx. pipiens lack high-sensitivity olfactory responsiveness to the host-attractant, lactic acid (Bowen et al.,

1988; Bowen, 1990), suggesting that the lack of blood feeding is associated with a shut- down of the host-seeking response. Thus, the arrest in ovarian development observed in diapausing Cx. pipiens is normally accompanied by a halt in blood feeding until diapause has been broken (Swellengrebel, 1929; Washino, 1977). Like most adult diapauses

(Denlinger, 1985), the diapause of Cx. pipiens appears to be the consequence of a shut- down in juvenile hormone synthesis by the corpora allata (Spielman, 1974; Readio et al.,

1999).

The hypertrophy of the fat body and elevation of lipid reserves that are associated with diapause are linked to a boost in sugar feeding that accompanies the entry into

52 diapause. The accumulation of lipid reserves occurs after adult eclosion; within a week,

females programmed for diapause by short daylength accumulate twice as much lipid as

their nondiapause counterparts, and these reserves are largely depleted during the course

of the winter (Mitchell and Briegel 1989a).

Although the physiological and ecological aspects of blood and sugar feeding in diapausing Cx. pipiens have been well described, the molecular events that contribute to

this metabolic decision have not been explored. In this study, we isolate and sequence three clones encoding digestive enzymes by suppressive subtractive hybridization: fatty

acid synthase, trypsin, and chymotrypsin-like serine protease, and then use these clones

to probe the metabolic pathways associated with the decision by the mosquito to enter

and terminate diapause. Since diapause in Cx. pipiens is programmed by both short

daylength and low temperature, we also distinguish between temperature and

photoperiodic effects. We conclude that the short-day programming of diapause results

in the downregulation of the genes encoding the blood digestive enzymes and the

upregulation of a gene associated with lipid sequestration.

MATERIALS AND METHODS

Insect Rearing

An anautogenous colony of Cx. pipiens L. was established in September, 2000,

from larvae collected in Columbus, Ohio (Buckeye strain). The colony was maintained

at 25°C, 75% r.h., with a 15hL:9hD daily light:dark cycle. Eggs and 1st instar larvae

were kept under colony conditions until the 2nd instar, and at that time larvae were either

53 kept in the colony rearing room (nondiapause, 25°C), moved to an environmental

chamber at 18°C, 75% r.h., and 15L:9D (nondiapause, 18°C), or placed in an

environmental room under diapause-inducing conditions of 18°C, 75% r.h., with a

9L:15D daily light:dark cycle (diapause, 18°C).

Larvae were reared in 18 x 28 x 5 cm plastic containers in de-chlorinated tap

water, fed a diet of ground fish food (TetraMin), and maintained at a density of ~250

mosquitoes per pan. Adults were kept in 30.5 x 30.5 x 30.5 cm screened cages and

provided constant access to water and honey-soaked sponges. Honey sponges were

removed from short-day cages 10-13 days after adult eclosion to mimic the absence of

sugar in the natural environment during the overwintering period. None of the

mosquitoes used in these experiments were offered a blood meal. To confirm diapause status, primary follicle and germarium lengths were measured and the stage of ovarian development was determined according to the methods described by Christophers (1911) and Spielman and Wong (1973).

Suppressive Subtractive Hybridization

Total RNA was isolated from pools of 20 females by grinding with 4.5 mm copper-coated spherical balls (“BB’s”) in 1 ml TRIzol® Reagent (Invitrogen). After homogenization, samples were spun at 12,404 g at 4°C for 10 min and the supernatant was used for RNA extraction following standard protocol (Chomczynski and Sacchi,

1987). RNA pellets were stored in absolute ethanol at -70°C and dissolved in 30 µl ultraPURE™ water (GIBCO) for use in cDNA synthesis (BD SMART™ PCR cDNA

54 Synthesis Kit, BD Biosciences) following standard protocol. Suppressive subtractive

hybridization (SSH) was performed using the Clontech PCR-Select™ cDNA Subtraction

Kit: the forward subtracted library was constructed using females in early diapause (7-10

days post adult eclosion, short daylength, 18°C) and the reverse subtracted library was

constructed using nondiapausing females (7-10 days post adult eclosion, long daylength,

18°C). Forward and reverse libraries were cloned using the TOPO TA Cloning™ Kit

(Invitrogen). Transformed plasmids were inserted into competent Escherichia coli cells

and grown overnight on Luria-Bertani (LB) plates containing X-Gal and ampicillin. For

each library, over 100 white colonies were isolated and grown overnight in LB-ampicillin

broth at 37°C. Colonies were then purified with QIAprep Spin Miniprep (QIAGEN) and

sequenced using the vector internal primer sites (T7 and M13R) at the Ohio State

University Plant-Microbe Genomics Facility on an Applied Biosystems 3730 DNA

Analyzer using BigDye® Terminator Cycle Sequencing chemistry (Applied Biosystems)

following manufacturer’s protocol.

Northern Blot Analysis

RNA was extracted from adults and pupae following the methods described

above. Pupae were sexed as females based on their large size and prolonged

development. Fifteen micrograms of denatured total RNA samples were separated by

electrophoresis on a 1.4% agarose denaturing gel (0.41 M formaldehyde,

1X MOPS-EDTA-sodium acetate). Visualization of ethidium bromide stained rRNA

under UV light exposure was used to confirm equal loading. Following the

55 TURBOBLOTTER™ Rapid Downward Transfer Systems protocol (Schleicher and

Schuell), the RNA was transferred for 1.5 hours onto a 0.45 micron MagnaCharge nylon

membrane (GE Osmonics) using downward capillary action in 3 M NaCl, 8 mM NaOH

transfer buffer, followed by neutralization in a 1 M phosphate buffer solution and UV crosslinking. The membrane was then air-dried and either stored at -20°C or used immediately for hybridization.

Digoxigenin (DIG)-labeled cDNA probes were developed from the three metabolically related genes generated in our forward and reverse subtracted SSH libraries. PCR was performed on each clone using the SSH nested primers (Clontech

PCR-Select™ cDNA Subtraction Kit) according to the following parameters: 94°C for 3 min and 35 cycles of 94°C for 30 sec, 60°C for 30 sec, and 72°C for 2 min, followed by a

7 min extension at 72°C and a 4°C hold. The PCR products were run on a 1% TAE agarose gel and the band of interest was excised from any remaining vector, extracted with Ultrafree®-DA (Millipore), and re-amplified by PCR. Fatty acid synthase, trypsin, and cymotrypsin-like serine protease cDNAs were individually labeled in an overnight

DIG reaction using 100ng of template DNA and the Dig High Prime DNA Labeling and

Detection Starter Kit II (Roche Applied Sciences). Probes were stored at -20°C.

Hybridization was carried out overnight followed by stringency washes and immunological detection using the Dig High Prime DNA Labeling and Detection Starter

Kit II (Roche Applied Sciences) according to manufacturer’s protocol. Blots were then exposed to chemiluminescence film (Kodak Biomax). Each northern was replicated three or more times. To confirm equal transfer of RNA, each membrane was stripped with 0.2

56 M NaOH/0.1% SDS and re-probed using DIG-labeled 28S cDNA, according to

manufacturer’s instructions.

3’ and 5’ RACE

The SMART™ RACE cDNA Amplification Kit (Clontech) was used to perform

both 5’ and 3’-rapid amplification of cDNA ends (RACE). For 3’ RACE, first strand

cDNA was synthesized from 5 µg total RNA using the manufacturer’s provided adaptor primer. Target cDNA was then amplified using the Universal Amplification Primer and a forward, gene-specific primer based on the sequence of the original clones: fatty acid

synthase (5’-AAT TAC GCC AAA CTG C AA GG-3’), trypsin (5’-CAA CTT CCT CTC

GTC CGG TA-3’), and chymotrypsin-like (5’GAT GAT CTG CCC AAG GAC TC-3’).

PCR consisted of a “hot start” at 94°C for 3 min and 35 cycles of 94°C for 30s, 60°C for

30s, and 72°C for 2 min, followed by an additional 7 min at 72°C.

The 5’ RACE was carried out using two gene specific reverse primers according

to manufacturer’s protocol. Template cDNA was synthesized with trypsin (5’-ACG TTG

GAG TAA ATT C-3’) and chymotrypsin-like (5’-TAT CAC AAC TCT TAA TTT C-3’)

reverse primers. After purification and TdT tailing of the cDNA, PCR was used to

amplify each gene using a second set of nested, reverse gene-specific primers and the

provided Abridged Anchor Primer. The PCR gene-specific primers were as follows:

trypsin (5’-GGT GAC CTC GCG ATC ATA GT-3’) and chymotrypsin-like (5’-TCC

CAA CCT TTT CGT TGA AG-3’). Two rounds of PCR were done according to the

parameters described above. The 3’- and 5’-RACE products were cloned and double-

57 sequenced as described above. Although several sets of gene-specific primers were used

in attempts to amplify the 5’ end of fatty acid synthase, full-length sequence was not

obtained.

Bioinfomatics Analyses

The 5’ and 3’ RACE products were edited and assembled using dnaLIMS

(dnaTools) and BioEdit Sequence Alignment Editor (Isis Pharmaceuticals). Similar sequences were identified by performing a BLASTn and BLASTx search in GenBank

(http://www.ncbi.nlm.nih.gov/). The deduced amino acid sequences were assembled, analyzed, and aligned with similar sequences using Blastp (NCBI), the Baylor College of

Medicine Search Launcher: Sequence Utilities (http://dot.imgen.bcm.tmc.edu/seq- util/seq-util.html), and BoxShade Server 3.21

(http://www.ch.embnet.org/software/BOX_form.html). Percent identities were obtained by blasting two sequences with the Blastp server using BLOSUM62 matrix in NCBI’s web server. The full-length nucleotide sequences for trypsin and chymotrypsin-like serine protease and the 3’ end of fatty acid synthase were deposited in GenBank and assigned accession numbers AY958426, AY958427, and AY958428, respectively.

RESULTS

Clone Identification

Three metabolically-related genes were identified by suppressive subtractive hybridization at the onset of diapause (7-10 days post adult eclosion): two blood digestive

58 enzymes in the class of serine proteases, trypsin and chymotrypsin-like serine protease,

were among the diapause-regulated genes that were putatively downregulated (obtained

from our reverse subtracted library), and fatty acid synthase was putatively diapause upregulated (obtained from our forward subtracted library). The cDNA matching trypsin is a 267 bp fragment matching with 96% identity the complete coding sequence of Cx. pipiens pallens trypsin mRNA. This portion of the clone corresponds to an 88 residue

deduced amino acid sequence that matches Cx. pipiens pallens amino acids 1 through 76.

The second clone, encoding a chymotrypsin-like gene, was also identified in the reverse

subtracted (downregulated) library. The chymotrypsin-like clone is 294 bp long and

encodes a deduced amino acid sequence of 97 residues, with 40% identity and 51%

positive matches to a serine protease from Anopheles gambiae. The forward subtracted

library (upregulated) yielded a 230 bp cDNA fragment with 85% identity to an Armigeres

subalbatus fatty acid synthase mRNA expressed sequence tag (EST). The deduced

amino acid sequence is 76 residues long and matches amino acids 2408 through 2468 in

fatty acid synthase from An. gambiae. The original clones of trypsin, chymotrypsin-like,

and fatty acid synthase were used to generate DIG-labeled probes for northern blot hybridization, producing bands of 0.9, 0.9, and ~8.0 Kb, respectively.

To obtain full-length cDNAs of trypsin, chymotrypsin-like, and fatty acid

synthase, the original SSH partial cDNA sequences were used to design gene-specific

primers for 5’ and 3’ RACE amplification. The resulting trypsin PCR products for 5’ and

3’ RACE were 397 and 501 bp, respectively. Both sequences overlapped the initial SSH

clone and yielded a total product size of 898 bp with a 783 bp open reading frame, as

59 shown in Figure 3.1. Our clone has a 46 and 27 base pair 5’ and 3’ untranslated region, respectively, with a putative polyadenylation signal (AATAAA) occurring at position

839. The deduced protein has an open reading frame starting at nucleotide 91 and is 261 residues long. Figure 3.2 shows the full-length sequence of the chymotrypsin-like cDNA, along with the predicted 240 residue amino acid sequence. The 5’ RACE yielded a 609 bp product, and 3’ RACE produced a segment 435 bp long. Together they form the complete chymotrypsin-like cDNA, which is 881 bp with an open reading frame of 720 bp. The 5’ untranslated region is 47 base pairs long, and the 3’ untranslated region is 94 bp with a polyadenylation site occurring at position 811.

The 3’ end of fatty acid synthase was obtained by RACE and resulted in a 954 bp clone that encodes 48 amino acids (Figure 3.3). Our clone has a large 3’ untranslated region 810 bp long. The putative polyadenylation signal was identified at position 906.

A Blastx search revealed that this segment includes a portion of the thioesterase domain, as determined in other known protein sequences (Holzer et al., 1989). Attempts at 5’

RACE were unsuccessful, thus the full-length sequence of fatty acid synthase was not obtained.

Comparison and Analysis of the Deduced Protein Sequences

The Cx. pipiens trypsin and chymotrypsin-like open reading frames include predicted mature active peptides in the family of serine proteases, identified by the histidine, aspartic acid, and serine residues that form the characteristic catalytic triad

60 (Walsh and Wilcox, 1970). The three cysteine bridges that form the disulphide bonds

essential for holding the polypeptide chains together are also conserved (Figures 3.4 and

3.5).

A multiple sequence alignment of the deduced trypsin amino acid sequence with

the sequences of other insect trypsins is shown in Figure 3.4. The Cx. pipiens trypsin

ORF shares 92% identity with trypsin from a close relative, Cx. pipiens pallens. When

compared with the well-described trypsins from Ae. aegypti, our sequence aligns closest with early trypsin (51%), compared to 31% identity with late trypsin. In addition, our trypsin aligns with 53% and 51% identities to an An. gambiae trypsin and Drosophila

melanogaster trypsin-like protease, respectively. The deduced amino acid trypsin sequence has a predicted activation peptide 16 amino acids long, assuming a signal peptide cleavage site after Gly-18 and a putative activation peptide cleavage site after

Lys-34. The putative signal peptide cleavage site was determined using the PSORT

program through the www at http://psort.nibb.ac.jp/form2.html (McGeoch, 1985; Von

Heijne, 1986), and the activation peptide cleavage site was determined from the highly

conserved IVGG sequence, common at the start of most active serine protease enzymes

(Yan et al., 1997; Muller et al., 1995). Our putative active chymotrypsin-like peptide,

however, begins with the tetrapeptide IFGG. The Asp residue characteristic of trypsin-

like serine proteases was also identified in Figure 3.4, along with the three residues that

make up the zymogen triad (Ser/His/Asp) that are involved in stabilization of the

proenzyme (Madison et al., 1993).

61 Our full-length clone encoding the second downregulated digestive enzyme is most similar to serine proteases with chymotrypsin activity, as indicated by a Blastp search. The serine protease ORF aligns most closely with an undescribed protein from the An. gambiae genome project with which it shares a 45% identity (Figure 3.5). A multiple sequence alignment highlights the identities with other insect serine proteases as follows: An. gambiae serine protease, 45%; An. darlingi chymotrypsin 1, 36%; and An. darlingi chymotrypsin 2, 37%. Using the PSORT program, we predicted the cleavage site of the signal peptide to be between Ala-20 and Arg-21. This would leave a dipeptide activation segment prior to the tryptic cleavage at Arg-22. In addition, the conserved Gly residue characteristic of chymotrypsin-like serine proteases (Kraut, 1977; Warshel et al.,

1989) is noted in Figure 3.5.

Our Cx. pipiens fatty acid synthase ORF is multi-aligned at the 3’ end with other fatty acid synthase sequences retrieved from GenBank (Figure 3.6). The Cx. pipiens fatty acid synthase deduced amino acid sequence is 80% identical to fatty acid synthase from

Ar. subalbatus and 65% identical to an undescribed expressed sequence tag from An. gambiae. In addition, our alignment shows 47% identity with the well-described fatty acid synthase from the chicken Gallus gallus and 37% identity with the chimpanzee Pan troglodytes. Our 3’ end also overlaps a portion of the thioesterase domain as described in

G. gallus (Holzer et al., 1989).

62 Confirmation of Diapause Up- and Downregulated Genes

Northern blot hybridizations confirmed the SSH results showing the downregulation of mRNA encoding trypsin and chymotrypsin-like and the upregulation of fatty acid synthase in early diapause (Figure 3.7). Our SSH comparison utilized mosquitoes reared under diapause-inducing (short daylength) and nondiapause–inducing

(long daylength) conditions at 18°C. The only environmental variable was daylength, and thus we can conclude that the distinctions we observed were in direct response to photoperiod. To further evaluate the role of temperature, we also used northern blots to compare nondiapausing mosquitoes reared at both 18°C and 25°C. The same results were observed at the two temperatures (Figure 3.7), thus the rearing temperature of the mosquitoes does not appear to be a primary environmental factor regulating the expression of fatty acid synthase, trypsin, and chymotrypsin-like serine protease.

Expression Patterns at the Onset of Diapause

The two blood digestive enzymes, trypsin and chymotrypsin-like, have identical patterns of mRNA expression in the early days following adult eclosion (Figure 3.8). In nondiapausing females, the transcripts were first detected by northern blot hybridization 2 days after adult eclosion, and a strong signal persisted from day 3 through to our final observation on day 7. This pattern is consistent with the onset of host-seeking behavior in our laboratory colony: females were fully ready to take a blood meal 2-3 days after adult eclosion. By contrast, neither trypsin nor chymotrypsin-like gene expression was detectable in the diapausing individuals at this early stage.

63 Fatty acid synthase, the gene encoding a key enzyme in the conversion of sugars

to fat, was expressed at a low level in nondiapausing females, and then only on days 2

and 3 after eclosion. By contrast, this gene was highly expressed in diapausing adults

beginning on day 3, and expression persisted throughout the remainder of the 7 day

observation period. In all six of the independent diapause replicates, the signal was

highest on day 4 and was reduced on day 5 and 6. This decrease in detectable message

varied in intensity, but it was consistently observed in all replicates on either day 5 or 6.

Thus, in diapausing females the onset of expression of fatty acid synthase occurred one

day later, but expression was higher and persisted longer than in nondiapausing females.

Expression Patterns throughout Diapause and at Diapause Termination

Expression of mRNAs encoding these three enzymes was also monitored

throughout diapause, beginning one week (7-10 days) post adult eclosion and then at 30-

day intervals thereafter for up to four months. Though trypsin was not expressed early in

diapause (Figure 3.8), a signal was evident by day 90 and persisted through day 120

(Figure 3.9). A similar pattern of expression was observed for chymotrypsin-like, but in

this case a weak signal was first noted on day 60. When diapause was broken at 2

months by transferring the females to long daylength and high temperature, both genes were highly expressed within one week (Figure 3.9).

Our northern blots showed that the mRNA encoding fatty acid synthase was highly expressed in diapausing females during the first week following adult eclosion

(Figure 3.8), but the expression was sporadic thereafter (Figure 3.9): the mRNA was

64 consistently undetectable on day 30, was strongly present on days 60 and 90, but was gone again on day 120. Expression was consistently high when diapause was broken.

DISCUSSION

Suppressive subtractive hybridization yielded three clones of potential interest for probing feeding responses in nondiapausing and diapausing individuals of Cx. pipiens.

Genes encoding two blood digestive enzymes, trypsin and chymotrypsin-like serine protease, are downregulated in early diapause, and fatty acid synthase, an enzyme involved in lipid sequestration, is highly upregulated at this time. We have confirmed these results by northern blot hybridization and have also demonstrated that the regulation of these genes is under photoperiodic control (short daylength) and not temperature. This is the first molecular evidence demonstrating that diapause-destined females are programmed to express a gene associated with the accumulation of lipid reserves and that these females have shut down the expression of genes associated with digestion of a blood meal.

We obtained the 3’ end of fatty acid synthase, which contains a deduced amino acid sequence that overlaps a portion of the thioesterase domain of fatty acid synthase in

G. gallus. As is typical of this gene, our clone also contains a long (807bp), 3’- untranslated region with a nucleotide sequence of low homology to the other known insect fatty acid synthase sequences. The translated region, however, is conserved

(>65%) among the three mosquito species examined. We have also identified full-length cDNA clones that encode trypsin and a chymotrypsin-like protein, two proteolytic blood

65 digestive enzymes in the class of serine proteases. Both deduced amino acid sequences contain the characteristic catalytic triad and the six cysteine residues typical of serine proteases (Wilcox, 1970). Our clones differ, however, in the residues involved in substrate specificity: the predicted active trypsin enzyme contains a negatively charged carboxylate (Asp 210) located at the bottom of the substrate binding pocket which is typical of trypsin-like serine proteases, while our second clone contains a hydrophobic substrate binding pocket (Gly-189) characteristic of chymotrypsin-like serine proteases.

Our trypsin clone also contains the three residues that make up the zymogen triad

(Ser/His/Asp) that is involved in stabilizing the inactive serine protease proenzyme

(Madison et al., 1993). However, our chymotrypsin-like serine protease contains only one of the three zymogen triad residues in the conserved location, a feature similar to a chymotrypsin-like protease from the human malaria vector An. gambiae (Han et al.,

1997). Our first serine protease clone thus appears to be a trypsin-like serine protease, and our second clone is most similar to a chymotrypsin-like serine protease.

The expression of these three genes investigated by northern blot hybridization revealed distinct patterns of expression at the onset of diapause, during four months in diapause, as well as at diapause termination. During the first seven days of adult life in diapause-destined females, fatty acid synthase is more highly expressed than in nondiapausing individuals and the expression persists for a longer period. These results are consistent with the pattern of sugar feeding in Cx. pipiens (Bowen, 1992): diapause- destined females fed on sugar more readily and for a longer period of time than their nondiapausing counterparts during the first 15 days of adult life. Although fatty acid

66 synthase is undetectable one month into diapause, it is sporadically expressed thereafter until diapause has been broken. In contrast, the genes encoding the blood digestive enzymes trypsin and chymotrypsin-like are completely “shut down” at the onset of diapause and remain downregulated until mid to late diapause, when females are preparing for diapause break.

In nondiapausing females, the upregulation of trypsin and chymotrypsin-like 2-3 days after adult eclosion corresponds with the expression patterns of chymotrypsin (Jiang, et al., 1997) and early trypsin (Kalhok et al., 1993; Noriega et al., 1996) in nondiapausing individuals of Ae. aegypti. Since our mosquitoes were not blood-fed, we would expect the trypsin we observed to be most similar to early trypsin in Ae. aegypti because early trypsin mRNA is abundant prior to blood feeding (Kalhok et al., 1993). The translation of early trypsin upon blood feeding is essential in activating the transcription of late trypsin, the major midgut endoprotease (Noriega et al., 1996). Indeed, our trypsin aligns most closely with early trypsin in Ae. aegypti (51%) , compared to 31% for late trypsin

(31%). In addition, our chymotrypsin-like clone is present prior to blood-feeding and is likely to be involved in blood digestion. Jian et al. (1997) characterized a female-specific chymotrypsin from Ae. aegypti and demonstrated the accumulation of mRNA 24 h after adult eclosion with translation being induced following blood feeding. Unlike early trypsin, chymotrypsin remained highly active during blood meal digestion in Ae. aegypti.

Our results suggest that it is unlikely that diapause-destined Cx. pipiens females take a blood meal prior to entering hibernation, since the molecular machinery necessary to process the blood meal is not functional in these individuals. The absence of trypsin

67 and chymotrypsin-like mRNA in diapause-destined Cx. pipiens is likely a result of a lack

in juvenile hormone (JH) at this time, since the transcriptional regulation of early trypsin

is under the control of JH in Ae. aegypti (Noriega et al., 1997). In Ae. aegypti, abdominal

ligations 1 h post adult eclosion led to a complete inhibition of early trypsin transcription

(Noriega et al., 1997). Instead of blood feeding, prehibernating females feed on nectar

and other plant products early in diapause and have the ability to convert the

carbohydrates into extra lipid reserves, a feature that is essential for survival throughout

the long winter. After 7 days of feeding on sugar, diapausing females accumulate nearly

twice as many lipid reserves as nondiapausing females reared at the same temperature

(Mitchell and Briegel, 1989a).

Once females enter diapause, fatty acid synthase continues to be expressed in mid to late diapause, thus females are likely to be capable of processing a sugar meal throughout diapause. This raises the possibility that Cx. pipiens may search for and

utilize a readily available sugar meal during the winter. This is plausible; several reports

have noted that diapausing females are active even in mid-winter and often leave their

hibernaculum during this time (Berg and Lang, 1948; Service, 1968; Buffington, 1972;

Onyeka and Boreham, 1987). We have also observed mid-winter movement in our field

sites and have observed that our laboratory-reared females will readily take a sugar meal

when honey-soaked sponges are placed into their cages 2-3 months after the onset

diapause (Robich et al., unpublished observations). An occasional sugar meal in mid-

winter would enable females to replenish lipid reserves and may enhance survival.

68 As the end of diapause approaches, the accumulation of trypsin and chymotrypsin- like mRNA indicates that females are preparing for blood feeding and subsequent egg production. This pattern follows the gradual increase of JH observed in diapausing females; by the end of winter JH titers reach levels equivalent to those observed in nondiapausing females (Readio et al., 1999). It is evident from the patterns of gene expression that regulation of fatty acid synthase, trypsin, and chymotrypsin-like is a part of the diapause program, and not due to other factors such as temperature, host availability, or feeding activities. It has been suggested that an occasional warm spell

(Indian Summer) in the fall may lead to Cx. pipiens taking a blood meal (Eldridge and

Bailey, 1979). But, previous behavioral data (Mitchell, 1983; Bowen et al., 1988;

Bowen, 1992), together with our current molecular evidence, suggest that diapause- destined females not only lack the host-seeking response but they are unable to process a blood meal. Instead, such females exposed to short daylength are programmed to feed on sugar and garner lipid reserves. This metabolic switch is thus a component of the diapause program.

REFERENCES

Bailey, C.L., Faran, M.E., Gargan, T.P., and Hayes, D.E. 1982. Winter survival of blood-fed and nonblood-fed Culex pipiens L. American Journal of Tropical Medicine and Hygiene 31:1054-1061.

Bellamy, R.E., Reeves, W.C., and Scrivani, R.P. 1958. Relationships of mosquito vectors to winter survival of encephalitis viruses. II. Under experimental conditions. American Journal of Hygiene 67:90-100.

Berg, M., and Lang, S. 1948. Observations of hibernating mosquitoes in Massachusetts. Mosquito News 8:70-71.