Impact of House Fly Salivary Gland Hypertrophy Virus ... - BioOne

VECTOR CONTROL, PEST MANAGEMENT, RESISTANCE, REPELLENTS

Impact of House Fly Salivary Gland Hypertrophy Virus (MdSGHV) on a Heterologous Host, Stomoxys calcitrans C. GEDEN,1 A. GARCIA-MARUNIAK,2 V. U. LIETZE,2 J. MARUNIAK,2

AND

D. G. BOUCIAS2

J. Med. Entomol. 48(6): 1128Ð1135 (2011); DOI: http://dx.doi.org/10.1603/ME11021

ABSTRACT The effect of Musca domestica salivary gland hypertrophy virus (MdSGHV) on selected Þtness parameters of stable ßies, Stomoxys calcitrans (L.), was examined in the laboratory. Virusinjected stable ßies of both genders suffered substantially higher mortality than control ßies. By day 9, female mortality was 59.3 ⫾ 10.1% in the virus group compared with 23.7 ⫾ 3.7% in the controls; mortality in virus-injected males was 78.1 ⫾ 3.1% compared with 33.3 ⫾ 9.3% for controls. Fecundity of control ßies on days 6 Ð9 was 49 Ð54 eggs deposited per live female per day (total, 8,996 eggs deposited), whereas virus-injected ßies produced four to Þve eggs per female on days 6 Ð7 and less then one egg per female per day thereafter (total, 251 eggs). Fecal spot deposition by virus-injected ßies was comparable to controls initially but decreased to ⬇50% of control levels by day 4 after injection; infected ßies produced only 26% as many fecal spots as healthy ßies on days 6 and 7. None of the virus-injected stable ßies developed symptoms of salivary gland hypertrophy. Quantitative real-time polymerase chain reaction demonstrated virus replication in injected stable ßies, with increasing titers of virus genome copies from one to four days after injection. MdSGHV in stable ßies displayed tissue tropism similar to that observed in house ßy hosts, with higher viral copy numbers in fat body and salivary glands compared with ovaries. Virus titers were ⬇2 orders of magnitude higher in house ßy than in stable ßy hosts, and this difference was probably due to the absence of salivary gland hypertrophy in the latter species. KEY WORDS stable ßy, houseßy salivary gland hypertrophy virus, polymerase chain reaction

The stable ßy, Stomoxys calcitrans (L.), is one of the most injurious pests of livestock and horses throughout the world. Severe ßy pressure results in diminished animal productivity and comfort, and there is increasing recognition of the role that stable ßies may play in movement of food-borne and other pathogens (Mramba et al. 2007, Castro et al. 2010). Changes in husbandry practices such as the widespread use of large round hay bales have resulted in substantial increases in stable ßy breeding and biting pressure in some areas (Talley et al. 2009). Traditional methods of chemical control often provide unsatisfactory results, and this has generated exploration for novel alternative stable ßy management tools. Pupal parasitoids have been used with some success (Skovgaard and Nachman 2004), a variety of traps are available (Taylor and Berkebile 2006), and insecticide-treated visual targets may provide a promising alternative to other insecticidal approaches (Foil and Younger 2006, Beresford and Sutcliffe 2010). With the exception of Entomophthorales fungal pathogens (Skovgaard and Steenberg 2002), little is known about pathogens of stable ßies. 1 Corresponding author: USDAÐARS, Center for Medical, Agricultural and Veterinary Entomology, 1600 SW 23rd Dr., Gainesville, FL 32608 (e-mail: [email protected]). 2 Department of Entomology and Nematology, 970 Natural Area Dr., Gainesville, FL 32611.

Musca domestica salivary gland hypertrophy virus (MdSGHV) is one of three known members of the newly described virus family Hytrosaviridae; the other two viruses infect adult tsetse (Glossinidae) and narcissus bulb ßies (Syrphidae) (Abd-Alla et al. 2009). MdSGHV has been found in house ßy populations globally, generally at fairly low prevalence rates (Geden et al. 2008, Prompiboon et al. 2010). Infected ßies typically display greatly enlarged salivary glands, and female ßies are rendered sterile by the viralinduced down-regulation of vitellogenesis (Lietze et al. 2007). In a survey of ßies that occur sympatrically with house ßies, no stable ßies were symptomatic for salivary gland hypertrophy, and injection of salivary gland homogenates from Þeld-collected stable ßies did not produce hypertrophy in house ßies (Geden et al. 2011). However, stable ßies that were injected with MdSGHV showed higher mortality than controls and displayed reduced ovarian development (based on dissection and scoring of ovarian condition). Moreover, extracts of salivary glands and ovaries from MdSGHV-injected stable ßies were infective for house ßies, suggesting viral replication in this heterologous host (Geden et al. 2011). The objectives of this study were to document the impact of MdSGHV on stable ßy Þtness, to compare viral replication in house ßies, Musca domestica L., and stable ßies, and to determine

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GEDEN ET AL.: HOUSE FLY SALIVARY GLAND HYPERTROPHY VIRUS

whether the virus replicates in the salivary glands, ovaries and fat body of stable ßies, tissue types that support replication in infected house ßies (Lietze et al. 2011). Materials and Methods Insect Rearing. House ßies were obtained from a colony that was originally established in the late 1950s from ßies collected near Orlando, FL. Larvae were reared on a mixture of wheat bran, alfalfa (Medicago sativa L.), cornmeal, and water (Hogsette 1992). Adult ßies were given water and a mixture of nonfat powdered milk, granulated sucrose, and powdered egg. Stable ßies were acquired from a colony established in the 1970s, and larvae were reared on the stable ßy medium described above. Adult stable ßies were given citrated cowÕs blood on soaked cotton pads that were changed daily. Pupae of both species were separated from larval media by water ßotation and were dried in an apparatus designed for this purpose (Bailey 1970). Virus Preparation and Inoculation. The MdSGHV3 genotype (Prompiboon et al. 2010) was prepared for injection using methods described in Lietze et al. (2007). In brief, salivary glands from Þve virus-infected house ßies were Þrst placed in 50 ␮l of sterile water, homogenized, vortexed, and water was added to bring the volume to 500 ␮l. The suspension was centrifuged for 3 min at 700 ⫻ g and Þlter-sterilized by centrifuging the supernatant a second time through a 45-␮m membrane for 5 min at 8,000 ⫻ g, and aliquots were stored at ⫺35⬚C until used for injections. For all experiments, young (⬍24-h-old) ßies were immobilized by cold and injected with 2.5 ␮l each of either sterile saline (controls) or MdSGHV preparations containing 10⫺4 infected gland pair equivalents (IGE) per microliter. Impact of MdSGHV on Stable Fly Fitness. One hundred ßies (50 each females and males) were injected with either virus or saline per replication; three replications were conducted using different cohorts of ßies and batches of virus. After injection, ßies were transferred to clean cages at 25⬚C and given a 10% sucrose solution for 24 h after injection. On days 2Ð 8 after injection each cage was provisioned with a cotton ball soaked in blood that was placed in a black nylon stocking in a cup. Fly mortality (males and females) was recorded daily until day 9. Blood cups were changed daily and eggs were rinsed from the stocking and counted. Egg deposition per live female was calculated daily for 9 d. During the above-mentioned experiment, we noticed that fecal spot deposition was lower in the virusinjected groups than in the controls. To document this effect, a second set of tests was conducted in which groups of 100 ßies were injected, fed and monitored daily for mortality as before. Beginning on day 1, a 9-cm-diameter disk of Þlter paper was placed in the bottom of each cage and changed daily for 7 d. Fecal spots were counted and the number of fecal spots deposited on the paper per live ßy each day was

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calculated. We estimated that the spots deposited on the Þlter paper disks represented ⬇25% of the total spots deposited by the ßies. Histology. A series of saline- and virus-injected ßies were dissected to determine whether MdSGHV can induce salivary gland hypertrophy, cause detectable perturbations in the reproductive tracts of male and female stable ßies, or both. Selected tissues (salivary glands, ovaries, and abdominal fat body tissue) were prepared for electron microscopy according to the Þxation and embedding protocols described in Lietze et al. (2009). Stained thin sections (90 Ð95 nm) were examined at 75 kV with an H-600 electron microscope (Hitachi, Tokyo, Japan). Titration of MdSGHV in Treated House Flies and Stable Flies. For both M. domestica and S. calcitrans one control and three virus-treated ßies were collected 0, 1, 2, 3, and 4 d postinjection (dpi) and frozen at ⫺70⬚C. Total DNA was extracted from individual ßies using the Masterpure Yeast DNA PuriÞcation Kit (Epicenter Technologies, Madison, WI) modiÞed to remove RNA from the samples by adding 2 ␮l of RNase A (5 ␮g/␮l) to 300 ␮l of lysis solution after insect maceration and incubating at 37⬚C for 30 min. After this step, the manufacturerÕs recommendations were followed. DNA concentrations were measured with a NanoDrop 1000 spectrophotometer (Thermo Fisher ScientiÞc, Waltham, MA), and 1 ␮l was electrophoresed on a 0.8% agarose gel to ensure RNA digestion. The presence of MdSGHV DNA in both S. calcitrans and M. domestica was assessed initially by conventional polymerase chain reaction (PCR) by using the SGHVenv primer pair and conditions outlined by Lietze et al. (2009) to detect a 487-bp region of the open reading frame (ORF) 47 (Garcia-Maruniak et al. 2008). PCR products were electrophoresed on 1.5% agarose gels and stained with ethidium bromide. To quantify numbers of viral genome copies in ßies at different times postinjection DNA preparations were subjected to quantitative real-time PCR (qPCR) in duplicate reactions as described in detail by Lietze et al. (2009), by using the qSGHVenv primer pair (Lietze et al. 2011). In brief, each 20-␮l reaction contained 10 ng of template DNA, 10 pmol of each primer, and 10 ␮l of 2⫻ SensiMix Plus SYBR ⫹ ßuorescein (Quantace, Norwood, MA) and was performed in an iCycler MyiQ Single Color Real Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA). A standard curve with serial dilutions of known MdSGHV genome copy numbers was included to calculate the numbers of genome copies present in each reaction and ultimately in each ßy. For this, the average viral copy number (from the duplicate readings) obtained per sample (corresponding to 10 ng of template DNA) was multiplied by the total amount of DNA that was extracted from each ßy. Replication of MdSGHV in Salivary Glands, Fat Body, and Ovarian Tissues of Stable Flies. Due to the observed impact of MdSGHV on ovarian development, studies were conducted to examine virus replication in the ovaries and two tissues that did not display pathological effects, the salivary glands and

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90 80

Healthy male

Infected male

Healthy female

Infected female

70 % morrtality

60 50 40 30 20 10 0 1

2

3

4

5

6

7

8

9

Days postt iinjection j ti

Fig. 1. Cumulative mortality of stable ßies after injection with either sterile saline or 2.5 ␮l of a solution containing MdSGHV at 10⫺4 infected gland pair equivalents per microliter.

abdominal fat body. In two replicate assays, female ßies were dissected at 7 dpi to obtain a total of two control and four viremic samples per tissue type. For each individual sample, tissues from two females were pooled. DNA was extracted and subjected to qPCR as described above for MdSGHV titration. In this assay, three different primer pairs speciÞc for MdSGHV ORFs 47, 86, and 106 (Lietze et al. 2011) were used. Transcription of MdSGHV Genes in Infected Tissues of Stable Flies. Transcription of Þve different MdSGHV genes was assessed for ovarian and fat body tissues of female stable ßies at seven dpi. For each replicate sample (one control, three infected), the ovaries and fat body from four ßies were pooled into separate microcentrifuge tubes containing 500 ␮l of Tri-Reagent (Thermo Fisher ScientiÞc) and subjected to RNA extraction as described by Lietze et al. (2011). To ensure total degradation of DNA from the RNA samples, DNase-treated samples were subjected to conventional PCR by using virus- and host-speciÞc primer pairs. DNA-free RNA samples were dispensed in 1-␮g aliquots and Þrst strand cDNA was synthesized using SuperScript VILO cDNA Synthesis Kit (Invitrogen, Carlsbad, CA). Relative transcript abundance was then measured by qPCR by using MdSGHV-speciÞc primer pairs targeting the viral ORFs 1, 10, 47, 86, and 106, and 10 ng of reverse-transcribed RNA (cDNA) per sample as template (Lietze et al. 2011). The 28S forward primer originally synthesized for M. domestica (Mdv28sFDQ656974, Lietze et al. 2011) and a S. calcitrans-speciÞc reverse primer (qSc28S-R, Table 1.

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5⬘-TTGAATAAACTTAAGCCAATG-3⬘), synthesized from GenBank EF531151 sequence information (Petersen et al. 2007) for a product size of 159 bp, were used to normalize threshold cycle (Ct) values obtained with viral primers ensuring that equal amounts of cDNA template were compared in all treatments (Pfafß 2001). Statistical Analyses. Daily egg and fecal spot deposition data were analyzed by one-way analysis of variance (ANOVA; virus-injected versus controls) by using the ANOVA procedure of the Statistical Analysis System (SAS) for Windows, release 8.02 (SAS Institute, Cary, NC). Viral genome copy numbers were log10 transformed and subjected to two-way ANOVA using the general linear model (GLM) procedure of SAS; means were separated using TukeyÕs studentized range test (Ko¨ hler et al. 1992, Cody and Smith 2006). Untransformed numbers were presented as means ⫾ SE. The normalized Ct values of viral transcripts (i.e., the Ct value obtained for each ORF after subtraction of the Ct value obtained for the 28S host reference gene) also were analyzed by GLM and TukeyÕs test.

Results and Discussion Effects of MdSGHV on Stable Fly Fitness. Virusinjected stable ßies of both genders showed substantially higher mortality than control ßies, and this effect was noticeable beginning on day 3 after injection (Fig. 1). By day 9, female mortality was 59.3 ⫾ 10.1% compared with 23.7 ⫾ 3.7% in the control females (F ⫽ 12.68; df ⫽ 1, 4; P ⬍ 0.05). Male mortality on day 9 was 78.1 ⫾ 3.1% compared with 33.3 ⫾ 9.3% for control males (F ⫽ 20.69; df ⫽ 1, 4; P ⬍ 0.05). At the onset of oviposition on day 5, fecundity of control ßies, measured by egg production, was ⬇20 times higher (20 eggs per live female) than for virus-injected ßies (one egg per live female; Table 1; Fig. 2A). Fecundity of control ßies on days 6 Ð9 was 49 Ð54 eggs deposited per live female, whereas virus-injected ßies produced four to Þve eggs per female on days 6 Ð7 and less than one egg per female thereafter. In its primary host, the house ßy, MdSGHV-infection completely inhibits ovarian development (Lietze et al. 2007). A related virus, GpSGHV, infecting tsetse ßies (Glossina spp.), also signiÞcantly reduces fecundity of its female hosts. For example, infected Glossina morsitans centralis Machado females had longer pregnancy cycles and produced pupae with lower weights

Fecundity of healthy stable flies and flies injected with MdSGHV Mean (SE) total no. eggs deposited on day

Mean (SE) no. eggs/live female on day

Day after injection

Healthy

Virus-injected

ANOVA Fa

Healthy

Virus-injected

ANOVA Fa

4 5 6 7 8 9

0 831(207) 2,187 (240) 1,910 (232) 2,057 (360) 1,987 (392)

0 21 (20) 114 (32) 95 (37) 10 (5) 10 (10)

15.1* 73.1** 59.9** 32.2** 52.0**

0 20 (5) 54 (8) 49 (5) 54 (10) 54 (16)

0 1 (1) 5 (2) 4 (2) 0.5 (0.4) 0.3 (0.3)

14.6* 35.3** 72.3** 29.8** 44.2**

a

Asterisks indicate signiÞcant differences between healthy and virus-injected ßies; *, P ⱕ 0.5; **, P ⱕ 0.01, df ⫽ 1, 4.

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Fig. 2. Reduction of fecundity by MdSGHV infection observed in S. calcitrans. (A) Eggs deposited during 24 h by stable ßies injected with saline (left) or MdSGHV (right) on day 5 postinjection. At 5 dpi, ovaries of a dissected healthy female (B) contain well developed eggs, whereas ovaries of a virus-challenged female (C) are comprised of undeveloped eggs; note the lack of hypertrophied salivary glands. (Online Þgure in color.)

than did their healthy counterparts (Sang et al. 1998). In a previous study on MdSGHV host range (Geden et al. 2011), we observed that ovarian development of stable ßies was suppressed by injection of a Danish isolate of MdSGHV, but this assessment was conducted by dissecting ßies and scoring ovarian condition on day 5. In the current study, we found that an initial cohort of 50 healthy females deposited a total of 8,996 eggs compared with 251 eggs among virustreated ßies. If a suitable delivery method could be developed for infecting ßies in the Þeld, MdSGHV Table 2.

could have a profound impact on stable ßy population dynamics, especially if it were introduced early in the ßy season. Stable ßies feed primarily on blood, but they also visit ßowers (Taylor and Berkebile 2008), and there is a growing knowledge base of factors that attract females to oviposition sites and host odors (Romero et al. 2006; Jeanbourquin and Guerin 2007a,b). House ßies are more readily infected with MdSGHV by surface contamination than by ingestion (Geden et al. 2011). If stable ßies are also susceptible to infection by contact, it may be possible to develop an attract-

Fecal spot deposition by healthy stable flies and flies injected with MdSGHV Mean (SE) no. fecal spots produced/live ßy on day

Day after injection

Healthy

Virus-injected

ANOVA Fa

2 3 4 5 6 7

3.9 (0.5) 7.1 (0.7) 10.4 (0.2) 14.1 (1.8) 11.3 (1.2) 13.7 (1.3)

3.1 (0.6) 3.1 (0.5) 4.8 (1.1) 4.2 (0.8) 3.0 (0.2) 3.6 (0.6)

1.22 ns 23.92** 25.01** 25.10** 48.84** 52.05**

Ratio, virus/control 0.80 0.44 0.46 0.30 0.26 0.26

a Asterisks indicate signiÞcant differences between healthy and virus-injected ßies; ns, not signiÞcant, P ⬎ 0.05; *, P ⱕ 0.5; **, P ⱕ 0.01, df ⫽ 1, 4.

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Fig. 3. Transmission electron micrographs of thin sections of ovarian tissue dissected from MdSGHV-challenged S. calcitrans. (A) Viremic epithelial cell associated with the outer layer of the infected ovary. Higher magniÞcation of the nucleus (B) reveals a dark staining virogenic stroma in the center and a less dense peripheral zone (scale bars in A and B ⫽ 2 ␮m). (C) Nucleocapsids (arrows) associated with the nuclear membrane; note the presence of nonenveloped particles in both nucleus and cytoplasm. (D) Virus particles within membrane bound vesicles (arrows) in the cytoplasm (scale bars in C and D ⫽ 500 nm).

and-infect strategy for stable ßies that exploit ßoral and oviposition site semiochemicals. The cause of higher mortality in virus-infected ßies is unclear, but data on fecal spot deposition indicate that infected ßies feed less or have difÞculty processing the bloodmeal. Fecal spot deposition by healthy ßies rose from 3.9 spots per live ßy per day on day 2Ð10-14 spots on days 4 Ð7 (Table 2). Spot deposition by virus-injected ßies was comparable to controls on day 2 but dropped to ⬇50% of control levels by day 4; infected ßies produced only 26% as many fecal spots as healthy ßies on days 6 and 7 (Table 2). The results suggest that ßy avidity for blood-feeding is greatly reduced among ßies who survive infection. It should be noted that Lietze et al. (2007) reported that MdSGHV-infection caused decreased levels of midgut proteolytic activity and suggested that the viral infection reduced the digestive capabilities of house ßies. Impaired protein digestion may in part explain the suppression of ovarian development observed in both insects. In our assays, ßies had free access to liquid citrated beef blood on soaked pads. This may have masked further impairment of salivary gland function of infected ßies that would only have been manifested if they had been fed on live hosts. Viral Replication. Dissection of more than one hundred MdSGHV-challenged stable ßies revealed that salivary glands did not display hypertrophy symptoms that characterize replication of this virus in its homologous host M. domestica (Lietze et al. 2011). Under the dissecting microscope, salivary glands from virus-in-

jected stable ßies were identical to glands dissected from control ßies. The only tissue that displayed gross changes was the ovary that in virus-challenged stable ßies lacked developed eggs (Fig. 2B and C). These Þndings suggest that the virus-induced female sterility is due to pathology in tissues other than salivary glands. Recent Þndings by Lietze et al. (2011) that MdSGHV is capable of replicating in non salivary gland tissues of house ßies support this contention. Electron microscopy of stable ßy tissues provided clear evidence of viral morphogenesis in cells comprising the connective tissue, including trachea and muscles, surrounding ovarian tissues (Fig. 3). Nucleocapsids measuring ⬇600 nm in length, characteristic of MdSGHV (Garcia-Maruniak et al. 2008), were observed to be produced in partially swollen nuclei of these associated cells and seemed to exit the nuclear envelope. No viral particles were observed in the nu-

Fig. 4. PCR ampliÞcation of DNA extracted from individual S. calcitrans injected with 2.5 ␮l of MdSGHV viral homogenate corresponding to 10⫺4 infected gland pair equivalents per microliter (V) or with saline solution as controls (C). Flies were collected either immediately after (0) or 1Ð4 dpi, and PCR was performed using the MdSGHV ORF 47 primers and 1 ␮l of undiluted template as speciÞed in Materials and Methods.

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Fig. 5. Viral replication of MdSGHV in S. calcitrans (Sc) and M. domestica (Md). The number of viral genomes copies was plotted per total insect. Data were collected immediately after injection (0 dpi) and daily until 4 dpi. Bars represent the standard error obtained from four biological replicates per sampling time.

clei of the salivary gland cells, fat body cells, or the follicle and nurse cells of the ovaries. However, like the situation found in viremic house ßies (Lietze et al. 2011), a limited number of trachea/tracheoles associated with the fat body, salivary gland, and ovarian tissues were observed to contain membrane-bound aggregates of rod-shaped virus particles. The undiluted DNA preparations from stable ßies injected with virus and extracted at 0, 1, 2, 3, and 4 dpi produced detectable MdSGHV PCR amplicons after 25 cycles by using the MdSGHV ORF 47 speciÞc primers (Fig. 4). Based on stain intensity of the PCR products the viral levels in these ßies seemed to increase over time until 3 dpi, suggesting that virus replication occurred in this heterologous host. None of the DNA preparations extracted from saline-injected ßies at 0 Ð 4 dpi produced detectable amplicons (Fig. 4). Results from qPCR further demonstrated that MdSGHV was capable of replicating in the stable ßy when injected in its hemolymph (Fig. 5). The mean number of viral copies detected per stable ßy on day 0 was 4 ⫻ 106 viral copies and was comparable to that detected initially in the house ßy. The MdSGHV replicated faster and achieved higher titers in its primary house ßy host. At 1 dpi, the number of viral copies obtained in house ßies was comparable (⬇3 ⫻ 109 copies per ßy) to the highest number found in stable ßies at three dpi (⬇2 ⫻ 109). At 2 dpi, 1010 viral copies were detected in house ßies whereas only 2 ⫻ 108 viral copies were detected in stable ßies. Although titers were

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lower than in house ßies, the increase in the number of viral copies with time conÞrmed MdSGHV replication in stable ßies. Tissue Tropism and Viral Transcription. The genome of MdSGHV was capable of replicating in stable ßy ovaries, fat body, and salivary glands at an average rate of 1.5 ⫻ 105Ð1.5 ⫻ 106 viral copies per 10 ng of DNA isolated from each tissue at 7 dpi (Table 3). Within each tissue, the number of viral copies was similar for each ORF (Proc GLM, F ⫽ 0.47, df ⫽ 2, P ⫽ 0.6288), which was expected because all three are single copy genes (Garcia-Maruniak et al. 2008). Background levels in control samples never exceeded values corresponding to 5.0 ⫻ 101 viral copies (data not shown), demonstrating that the virus indeed replicated in the tested tissue samples. Similar to what was found in infected house ßies (Lietze et al. 2011) signiÞcantly higher viral copy numbers were obtained in stable ßy salivary glands and fat body in comparison to the ovaries (proc GLM, F ⫽ 94.27, df ⫽ 2, P ⬍ 0.001; P ⬍ 0.05, TukeyÕs test). However, although the number of viral copies per 50 ng of DNA found in house ßy salivary glands was more than double the number detected in its fat body (Lietze et al. 2011), there was no signiÞcant difference between those two tissues in the stable ßies (Table 3). It should be noted that all three tissue types are heavily tracheated and that only a minor portion of the associated tracheal system could be removed during dissections. The presence of viral genome copies in all examined tissues of viruschallenged stable ßies and house ßies (Lietze et al. 2011, this study), and our ultrastructural Þndings here support the notion that MdSGHV may replicate in cells of the tracheal system. Further comparison of the MdSGHV tissue tropism in the two host species revealed noticeable difference in the quantity of viral copies. The average number of MdSGHV copies measured in stable ßy tissues at seven dpi was 34, 32, and 51 times lower than in the ovaries, fat body, and salivary glands, respectively, of comparable amounts of DNA from infected house ßies at 3 dpi. Although MdSGHV infections of both house ßies and stable ßies reduce ovarian development and do not cause any visible symptoms in the fat body, infection causes hypertrophy in the salivary glands of the house ßies. The difference in viral titer between these two insects may be attributed to the lower level of replication and lack of hypertrophy in stable ßy salivary glands compared with house ßies. The massive virus proliferation that occurs in the hypertrophied house ßy glands (Lietze et al. 2007, 2011) probably

Table 3. Mean (ⴞSE) MdSGHV genome copy numbers detected in 10 ng of DNA from S. calcitrans ovaries, fat body, and salivary glands 7 d after virus injection Tissue

ORF47

ORF86

ORF106

Avg of three ORFsa

Ovaries Fat body Salivary glands

1.4E05 (2.6E04) 9.7E05 (9.6E04) 1.5E06 (4.5E05)

1.5E05 (3.8E04) 9.5E05 (1.2E05) 1.4E06 (4.2E05)

1.6E05 (2.1E04) 1.1E06 (1.3E05) 1.6E06 (3.9E05)

1.5E05 (1.5E04)a 1.0E06 (6.4E04)b 1.5E06 (2.2E05)b

Measurements were performed by qPCR targeting three different MdSGHV ORFs. a Mean copy numbers followed by different letters are signiÞcantly different (P ⬍ 0.05; TukeyÕs test).

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Table 4. Comparative MdSGHV gene transcription in ovaries and fat body of infected S. calcitrans measured by normalized threshold cycle (Ct) values during real-time PCR by using 10 ng of reverse-transcribed RNA (cDNA) and primers targeting five different viral ORFs MdSGHV ORF 1 10 47 86 106

Ct (mean of 3 samples) Ovaries

Fat body

17.57aA 18.03aA 17.59abA 15.73bA 18.14aA

14.61aB 13.86aB 13.44abB 12.32bB 15.40aB

Threshold cycle values followed by different letters are signiÞcantly different (P ⬍ 0.05; TukeyÕs test). Lowercase letters indicate differences between viral ORFs within each column, and uppercase letters indicate differences between the two tissues within each row.

accounts for much of the estimated difference in titer observed between these two ßy species. These Þndings, together with the results of the fecundity tests, support our contention that the salivary gland hypertrophy symptom observed in house ßies is not a requisite for female sterilization. Transcription of Þve different MdSGHV genes was conÞrmed in both the fat body and ovaries of infected stable ßies. The normalized Ct values presented in Table 4 show that in both tissues ORF 86 was transcribed at a signiÞcantly higher level than ORFs 1, 10, and 106, whereas transcript abundance of ORF 47 was intermediate but did not differ signiÞcantly from any of the other ORFs (Proc GLM, F ⫽ 3.46, df ⫽ 4, P ⫽ 0.0266; P ⬍ 0.05, TukeyÕs test). A similar trend was found in MdSGHV-infected house ßies tissues (Lietze et al. 2011), but the levels of transcription (determined by comparing normalized Ct valued between the two studies) were always lower in stable ßies than in house ßies. Another noteworthy similarity of MdSGHV transcription in these two insects is that in stable ßies all ORFs were transcribed at signiÞcantly higher levels in fat body tissue than in ovarian tissue (Proc GLM, F ⫽ 49.92, df ⫽ 4, P ⬍ 0.0001), a Þnding that may be related to the higher number of viral copies present in that tissue. The ORFs included in this study code for structural and nonstructural genes located throughout the viral genome (Garcia-Maruniak et al. 2008) and have been shown to produce transcripts in infected house ßies (Salem et al. 2009, Lietze et al. 2011). In summary, MdSGHV from house ßies was capable of replicating in the stable ßy, a heterologous host. Viral replication was demonstrated using a combination of ultrastructural and molecular analyses. Although infections did not result in salivary gland hypertrophy, a symptom characteristic of primary host infection with Hytrosaviridae, infected stable ßies exhibited female sterilization, another characteristic of infection by this group of viruses. Infection affected other Þtness parameters such as survival and the uptake or digestion of bloodmeals. From the standpoint of reducing biting pressure in ßy management programs, widespread infection with MdSGHV could result in the equivalent of a 75% reduction in ßy numbers. Because losses due to stable ßies have been

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estimated at ⬎$1 billion/yr (Taylor and Berkebile 2006) even a partial reduction in ßy biting pressure could bring signiÞcant economic beneÞts. The Hytrosaviridae currently represents a small group of recently discovered viruses; further work is needed to determine whether there are other as-yet undiscovered viruses associated with the higher Diptera. Acknowledgments We thank M. Doyle and H. Furlong for providing ßies for these studies and Karen Kelley (UF-ICBR EM, Gainesville, FL) for assistance during electron microscopy. Financial support was provided in part by USDA/NRI grant 200735302-18127.

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