A Wolbachia Symbiont in Aedes Aegypti Disrupts Mosquito Egg Development to a Greater Extent When Mosquitoes Feed on Nonhuman Versus Human Blood

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ARTHROPOD/HOST INTERACTION,IMMUNITY A Wolbachia Symbiont in Aedes aegypti Disrupts Mosquito Egg Development to a Greater Extent When Mosquitoes Feed on Nonhuman Versus Human Blood

1,2 1,3 1,4 CONOR J. MCMENIMAN, GRANT L. HUGHES, AND SCOTT L. O’NEILL

J. Med. Entomol. 48(1): 76Ð84 (2011); DOI: 10.1603/ME09188 ABSTRACT A vertebrate bloodmeal is required by female mosquitoes of most species to obtain nutrients for egg maturation. The yellowfever mosquito, Aedes aegypti (L.), feeds predominantly on humans, despite having the capacity to use blood from other hosts for this process. Here, we report that female Ae. aegypti infected with a virulent strain of the intracellular bacterium Wolbachia pipientis (wMelPop) from Drosophila melanogaster (Meigen) have a reduced ability to use blood for egg development. Blood feeding by wMelPop-infected females on mouse, guinea pig, or chicken hosts resulted in a near complete abolishment of reproductive output associated with both a decline in the numbers of eggs oviposited as well as the hatching rate of successfully laid eggs. In contrast, the reproductive output of wMelPop-infected females fed human blood was only mildly affected in comparison to individuals fed animal blood sources. Blood-feeding assays over two reproductive cycles deÞnitively illustrated a nutritional interaction between host blood source and egg development in wMelPop-infected Ae. aegypti. Removal of Wolbachia from mosquitoes using antibiotic treatment rescued egg development on all blood sources. Further investigation of this phenotype may provide new insights into the nutritional basis of mosquito anthropophily.

KEY WORDS Wolbachia, reproduction, mosquito, anthropophily

Female mosquitoes of most species must ingest a ver- fecundity of Ae. aegypti, compared with other verte- tebrate bloodmeal to obtain nutrients required for egg brate hosts (Scott et al. 1997, Costero et al. 1998, development (Attardo et al. 2005). The preference for Harrington et al. 2001, Braks et al. 2006). A greater human blood by a number of mosquito species (an- understanding of the nutritional basis of anthropoph- thropophily) aids in the transmission of human patho- ily may provide insight into important factors associ- gens such as dengue and malaria (Besansky et al. ated with host preference for humans, and potentially 2004). Aedes aegypti (L.), the principal vector of den- assist in the rational design of strategies that interfere gue, is a highly anthropophilic mosquito that lives in with this behavior. and around human habitation. Previous studies have A novel method for the control of dengue that has determined that Ae. aegypti feeds predominantly on been proposed involves the introduction of strains of humans in the Þeld (Scott et al. 1993b, Ponlawat and the obligate intracellular bacterium Wolbachia pipi- Harrington 2005), often taking bloodmeals multiple entis into Ae. aegypti populations to modify mosquito times within a single gonotrophic cycle (Scott et al. population age structure (Sinkins and OÕNeill 2000, 1993a, 2000). Cook et al. 2008) as well as directly modulate vector The nutritional components of human blood that competence (Moreira et al. 2009). Wolbachia are ma- differentiate it from other blood sources and subse- ternally transmitted endosymbionts that naturally in- quently contribute to the preference of Ae. aegypti for fect a wide diversity of invertebrates (Hilgenboecker humans are not fully understood (Harrington et al. et al. 2008), including many medically important mos- 2001). Blood feeding on humans has been shown to quito species (Sinkins 2004). Previously, we described increase energy reserves, survival rate, and lifetime the stable introduction of a virulent Wolbachia strain (wMelPop) from Drosophila melanogaster (Meigen) into Ae. aegypti (McMeniman et al. 2009), a species not 1 School of Biological Sciences, The University of Queensland, QLD naturally infected with Wolbachia. 4072, Australia. 2 Current address: Laboratory of Neurogenetics and Behavior, The One major physiological difference between D. Rockefeller University, 1230 York Ave., New York, NY 10065. melanogaster and Ae. aegypti is the requirement for the 3 Current address: The W. Harry Feinstone Department of Molec- latter species to blood feed to obtain nutrients for ular Microbiology and Immunology, Bloomberg School of Public oogenesis. In Drosophila, Wolbachia bacteria are Health and Malaria Research Institute, Johns Hopkins University, 615 North Wolfe St., Baltimore, MD 21205. thought to be dependent on their hosts for a range of 4 Corresponding author, e-mail: [email protected]. metabolites including amino acids, iron, and lipids

0022-2585/11/0076Ð0084$04.00/0 ᭧ 2011 Entomological Society of America January 2011 MCMENIMAN ET AL.: Wolbachia AND Ae. aegypti EGG DEVELOPMENT 77

(Wu et al. 2004, Brownlie and OÕNeill 2006, Brownlie Research, Animal Ethics Protocol P361). Written con- et al. 2009), which are also essential for mosquito egg sent was obtained from all human subjects used for development (Ziegler and Ibrahim 2001, Attardo et al. blood feeding. 2006, Zhou et al. 2007). Despite this potential conßict, To assess the ability of wMelPop-infected Ae. ae- wMelPop-infected Ae. aegypti are able to use blood gypti to use the blood from a wider range of host from humans, their preferred host, for egg develop- species for egg development, we conducted standard- ment with minimal costs to reproductive Þtness for ized membrane-feeding trials. For membrane feeding, young females (McMeniman et al. 2009). fresh whole mouse, chicken, and sheep blood antico- In this study, we examined the effect of wMelPop agulated with trisodium citrate (4 mg/ml), and deÞ- infection on the ability of this mosquito to use less brinated sheep blood, were purchased from a com- preferred nonhuman blood sources for egg develop- mercial supplier (IMVS Veterinary Services Division, ment. Here, we report that female Ae. aegypti infected Gilles Plains, SA, Australia). Anticoagulated (4 mg/ml with wMelPop have a drastically reduced ability to use trisodium citrate) and deÞbrinated pig blood were blood from nonhuman hosts for this process. We used sourced from a local abattoir (KR Castlemaine, blood from a wide variety of animal hosts, and anti- Toowoomba, QLD, Austrailia). Bloodmeals were pre- biotic clearance of Wolbachia to demonstrate an in- sented to premated 5-d-old females in glass membrane teraction between blood source and wMelPop infec- feeders (Rutledge et al. 1964) maintained at 37ЊC and tion that profoundly inßuences egg development in Þtted with Hemotek collagen membrane (Discovery Ae. aegypti. Workshops, Accrington, Lancashire, United King- dom). All bloodmeals were supplemented with 1 mM ATP to stimulate engorgement (Kogan 1990). Imme- Materials and Methods diately after a set feeding period of 30 min, unfed Mosquito Strains and Rearing. The following Ae. females were removed from treatment cages. aegypti strains were used in this study: 1) the In both laboratory animal and membrane feeding wMelPop-infected strains PGYP1 and PGYP2; 2) trials, sucrose sources were removed from females 5 h their uninfected tetracycline-cured counterparts before blood feeding. Postfeeding, blood-fed females PGYP1.tet and PGYP2.tet; and 3) the uninfected wild- were provided with constant access to 10% sucrose type strain JCU, from which PGYP1 and PGYP2 were solution and water until fecundity and fertility assays originally derived upon transinfection with wMelPop were done. (McMeniman et al. 2009). Mosquitoes were main- Blood-Feeding Trials: Human Volunteers. To ex- tained at 25ЊC, 75Ð85% RH, and a photoperiod of 12:12 amine variation in fecundity associated with blood (L:D) h. Larvae were reared at set density (150 larvae sources from different individual humans, PGYP1 and per 3 liters of distilled water in 30- by 40- by 8-cm PGYP1.tet mosquitoes were fed on eight human vol- plastic trays) and fed 150 mg of Þsh food (TetraMin unteers (four male and four female) in four replicate Tropical Tablets, Tetra, Melle, Germany) per pan experiments. Sucrose sources were removed5hbe- each day until pupation. Adults were kept in screened fore blood feeding and premated 5-d-old adult females 30- by 30- by 30-cm cages with ad libitum access to 10% mosquitoes were allowed to feed on exposed forearms sucrose solution and water. Five day old females were for twenty minutes (University of Queensland, Hu- provided with a human bloodmeal (University of man Ethics Approval 2007001379). In each of the four Queensland, Human Ethics Approval 2007001379) for replicates, the left and right arms were alternated for egg production. For routine colony maintenance, eggs the wMelPop-infected PGYP1 and tetracycline- from the PGYP1 and PGYP2 strains were hatched 5Ð7 treated PGYP1.tet mosquitoes. Postblood feeding, the d postoviposition (i.e., without prolonged desicca- sucrose source was returned. tion) to initiate the next generation. Fecundity and Egg Viability Assays. Females were Tetracycline Treatment. The tetracycline-cured isolated individually for egg-laying 96 h postblood PGYP1.tet and PGYP2.tet strains of Ae. aegypti were meal for a period of 48 h in plastic Drosophila vials generated at G8 and G11 posttransinfection by the containing wet Þlter paper as an oviposition substrate. addition of tetracycline (1 mg/ml) to the adult diet and Eggs were hatched 120 h after oviposition by submer- recolonized with resident gut microßora from wild-type sion in nutrient-infused deoxygenated water (75 mg larvae as described previously (McMeniman et al. 2009). TetraMin/liter) for 48 h. To hatch any remaining eggs, Proceeding tetracycline treatment, strains were al- oviposition papers were dried brießy and then resub- lowed to recover for at least two generations before mersed for a further 5 d before the Þnal numbers of being used in experiments. hatched larvae were recorded. Insemination of fe- Blood-Feeding Trials: Laboratory Animals and males was veriÞed by dissection of spermathecae in Membrane Feeding. In blood-feeding assays that used phosphate buffer saline followed by direct observa- live hosts, premated 5-d-old adult females were blood- tion of sperm by light microscopy. fed to repletion using a human forearm (University of Statistics. All statistical comparisons were con- Queensland, Human Ethics Approval 2007001379); ducted using Statistica, version 8 (StatSoft, Tulsa, OK). outbred Quackenbush mice anesthetized with a mix- Normality of experimental data was determined using ture of ketamine and xylazine (UQ Animal Ethics the KolmogorovÐSmirnov test. If data were not nor- Approval SIB/549/07/NIH); or guinea pigs anesthe- mally distributed, nonparametric tests (MannÐWhit- tized with Nembutal (Queensland Institute of Medical ney U [MWU], one-way KruksalÐWallis, and Wil- 78 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 48, no. 1

Fig. 1. Effect of rodent blood on egg development in the wMelPop-infected PGYP1 and naturally uninfected JCU Ae. aegypti strains. Females were blood fed at 5 d posteclosion on mice (A) or guinea pigs (B). The average number of unhatched and hatched eggs Ϯ SE for each treatment is shown, and the numbers of replicates for each strain are denoted in parentheses above error bars. Assays were conducted with G9 PGYP1 females. coxon matched pairs tests) were used to analyze data P Ͻ 0.001; n ϭ 30 females per strain). This decline in sets. StudentÕs t-test, analysis of variance (ANOVA), fecundity was accompanied by an approximate 120- and post hoc comparisons were used to analyze nor- fold decrease in egg viability from 83.7% in the JCU mally distributed data sets. strain to 0.7% in the PGYP1 strain (MWU, U ϭ 0.0, df ϭ 60, P Ͻ 0.001). These trends were similar to those obtained after blood feeding with guinea pigs with Results both fecundity and egg viability of PGYP1 signiÞcantly Egg Development on Rodent Blood. When females reduced relative to the JCU strain (MWU, P Ͻ 0.001 from the wMelPop-infected PGYP1 strain (hereafter for both comparisons; n ϭ 30 females per strain). In referred to as PGYP1) were provided with a mouse or general, eggs oviposited by PGYP1 females after ro- guinea pig bloodmeal a dramatic reduction of egg dent bloodmeals were less melanized and smaller than development was observed relative to females from those obtained after a human meal (Fig. 2). the uninfected wild-type JCU strain (Fig. 1). This was Egg Development in the Absence of Wolbachia. To associated with both a decline in the numbers of eggs determine whether the reduction in PGYP1 egg de- oviposited by each female and the percentage of velopment after a rodent bloodmeal was due to Wol- hatched larvae. For example, PGYP1 females that bachia or differences in host genetic background be- were blood fed on mouse blood produced signiÞcantly tween the PGYP1 and JCU strains, we cured the fewer eggs than JCU females (MWU, U ϭ 3.0, df ϭ 60, wMelPop infection from PGYP1 with tetracycline. Af-

Fig. 2. Phenotypic differences in embryo morphology after blood feeding of wMelPop-infected PGYP1 females on different blood sources. PGYP1 females were fed on human (A), mouse (B), or guinea pig (C) blood, and the resulting eggs were photographed 24 h postoviposition. Scale bar ϭ 0.5 mm. (Online Þgure in color.) January 2011 MCMENIMAN ET AL.: Wolbachia AND Ae. aegypti EGG DEVELOPMENT 79

Fig. 3. Tetracycline-induced restoration of egg development. Average number of unhatched and hatched eggs Ϯ SE resulting from wMelPop-infected PGYP1 and tetracycline-cured PGYP1.tet females (A); and PGYP2 females and PGYP2.tet females (B), after blood feeding on mice. Numbers of replicates for each strain are shown in parentheses above error bars.

Assays were conducted with G16 PGYP1 and G24 PGYP2, females respectively. ter antibiotic clearance of wMelPop, the ability of not signiÞcantly change either the total number of females from the resulting uninfected PGYP1.tet eggs oviposited or egg viability. Human blood resulted strain to use mouse blood for reproduction was fully in high fecundity and embryo viability for both cycles, restored (n ϭ 36 females per strain) (Fig. 3A). More- whereas mouse blood consistently decreased fecun- over, this was also the case after tetracycline-treat- dity and embryo viability. However, in the human- ment of a second wMelPop-infected Ae. aegypti strain, mouse treatment, a mouse bloodmeal on the second PGYP2, which served as an independent biological cycle resulted in a signiÞcant inhibition of both fe- replicate (n Ն 34 females per strain) (Fig. 3B). cundity (Wilcoxon matched pairs test, T ϭ 5.0, Z ϭ Nutritional Interaction Between Blood Source and 3.95, df ϭ 22, P Ͻ 0.001) and egg viability (T ϭ 1.0, Z ϭ Egg Development. To better characterize the nutri- 4.17, df ϭ 23, P Ͻ 0.001). The opposite effect was tional interaction between blood source and egg de- evident in the mouse-human treatment, where a hu- velopment in the PGYP1 strain, we blood fed cohorts man bloodmeal in the second cycle completely res- of PGYP1 females on either 1) a human volunteer or cued fecundity (T ϭ 16.0, Z ϭ 4.05, df ϭ 26, P Ͻ 0.001), laboratory mouse for two successive reproductive cy- and signiÞcantly restored egg viability (T ϭ 0.0, Z ϭ cles (i.e., humanÐhuman, mouseÐmouse) or 2) alter- 4.46, df ϭ 26, P Ͻ 0.001). nated the blood source provided for the second cycle Blood Source Host Range Effect. We investigated (i.e., humanÐmouse, mouseÐhuman) (Fig. 4). In hu- the host range of this phenotype using artiÞcial mem- manÐhuman and mouseÐmouse treatments, receiving brane-feeding trials with blood sourced from a variety the same blood source for two successive cycles did of common laboratory and agricultural animals. Stan-

Fig. 4. Nutritional interaction between blood source and egg development in the wMelPop-infected PGYP1 strain. (A) Average number of eggs produced per female Ϯ SE. (B) Average percentage of embryo hatch Ϯ SE, after blood feeding PGYP1 females for two successive reproductive cycles on either human or mouse blood, or alternating the blood sources between cycles. Bloodmeals for the Þrst cycle were given at 5 d posteclosion, and the bloodmeals for the second cycle at 11 d ϭ posteclosion (n 35 females per treatment). Assays were conducted with G24 PGYP1 females. 80 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 48, no. 1

Fig. 5. Effect of different blood sources on PGYP1 egg development. Average number of hatched and unhatched eggs Ϯ SE for wMelPop-infected PGYP1 and tetracycline-cured PGYP1.tet strains after blood feeding on anticoagulated chicken, mouse, pig, and sheep blood. Females were blood fed at 5 d posteclosion. The numbers of females (n) per strain for each treatment are shown in parentheses below the bars. Assays were conducted with G14 PGYP1 females. dardized comparisons of fecundity and egg viability 2.7%) blood were the worst blood sources affecting were made using the PGYP1 and tetracycline-cured embryo viability, followed by pig (41.3 Ϯ 7.2%) and PGYP1.tet strain that share a common genetic back- sheep blood (49.0 Ϯ 5.3%) that produced intermediate ground, and the following blood sources: anti-coagu- levels of hatch. lated chicken, mouse, pig, and sheep blood (n Ն 10 No signiÞcant differences in fecundity or egg via- females per strain per treatment) (Fig. 5). In addition, bility were found between groups of PGYP1 females we also evaluated the effect of deÞbrination and an- fed on either deÞbrinated or anticoagulated pig and ticoagulation processes on egg development, by in- sheep blood (MWU, P Ͼ 0.05 for all comparisons). The cluding deÞbrinated pig and sheep blood in these trials same trend was also evident in comparisons between for comparison (n Ն 25 females per strain per treat- anticoagulated mouse blood and results using anes- ment; data not shown). thetized mice, where effects on PGYP1 fecundity and Consistent with earlier observations, a signiÞcant egg viability were not statistically different from one interaction between blood source and infection status another (fecundity: t-test, t ϭϪ0.36, df ϭ 47, P ϭ was determined to affect fecundity in membrane feed- 0.719; and viability: MWU, U ϭ 229.0, df ϭ 49, P ϭ ϭ Ͻ Ն ing trials (ANOVA, F5,312 5.99, P 0.001). On all 0.139; n 20 females per strain). blood-sources, individual pairwise comparisons re- Inﬂuence of Human Host on Fecundity. To exam- vealed the numbers of eggs oviposited by PGYP1 fe- ine whether the individual human host used for blood males were signiÞcantly reduced relative to PGYP1.tet feeding inßuenced the fecundity of wMelPop-in- females (TukeyÕs honestly signiÞcant difference post fected Ae. aegypti, we fed PGYP1 and PGYP1.tet (n ϭ hoc test, P Ͻ 0.001 for all comparisons). The same 35 females per treatment) on eight different volun- trend also was observed for egg viability, with signif- teers, both male and female, and examined subsequent icant differences in embryo hatch rates detected be- egg production. This experiment was replicated four tween PGYP1 and PGYP1.tet strains fed chicken, times for each volunteer and mean fecundity is re- mouse, pig, and sheep blood (MWU, P Ͻ 0.001 for all ported in Fig. 6. The presence of Wolbachia resulted comparisons). in a signiÞcant reduction in the fecundity of mosqui- Mean hatch rates of PGYP1.tet eggs were consis- toes relative to the tetracycline-cured controls ϭ Ͻ tently high, exceeding 85% on all blood sources. In (ANOVA, F1,1584 74.21; P 0.001). A signiÞcant contrast, embryo hatch rates varied signiÞcantly ac- interaction between human host and infection status cording to blood source received by PGYP1 females was also determined to affect fecundity (ANOVA, ϭ Ͻ ϭ Ͻ (KruksalÐWallis, H5,171 101.93, P 0.001). Overall, F7,1584 7.92; P 0.001). The sex of the human host chicken (0.4 Ϯ 0.2% [SE] hatch) and mouse (4.8 Ϯ did not inßuence fecundity. January 2011 MCMENIMAN ET AL.: Wolbachia AND Ae. aegypti EGG DEVELOPMENT 81

Fig. 6. Effect of human host on fecundity of the wMelPop-infected PGYP1 and tetracycline-cleared PGYP1.tet strains. Average number of eggs Ϯ SE for cohorts of female mosquitoes (5 d posteclosion) blood fed on different human volunteers. Volunteers 1Ð4 and 5Ð8 were male and female, respectively. Each bar represents the mean of four replicate experiments with n ϭ 35 mosquitoes in each trial. Replicate experiments were conducted with PGYP1 females in consecutive generations from

G29 to G33 posttransinfection.

Discussion inbreeding effects in this strain that accentuate the physiological costs of this bacterial infection. Never- This study provides evidence for a nutritional in- theless, the inclusion of tetracycline-treated mosquito teraction between wMelPop infection and host blood controls in each experiment allowed us to make quan- that inßuences Ae. aegypti egg development. Both mosquito fecundity and egg viability were severely titative comparisons between blood sources within a impacted when wMelPop-infected females fed on cer- mosquito generation, and qualitative comparisons be- tain nonhuman blood sources, and, in some cases, this tween mosquito generations. phenotype was associated with gross defects in em- In general, the magnitude of fecundity costs when bryo morphology. These results were unexpected PGYP1 females fed on human blood (18Ð37% reduc- when considered that Ae. aegypti is normally able to tion in relative fecundity) was less than that seen thrive on a wide variety of nonhuman vertebrates in when mosquitoes were fed on different animalsÕ the lab (Woke 1937, Bennett 1970), including all of the blood, and a continuum of host effects was observed. blood sources used in this study. Consistent with the For example, fecundity costs of up to 68% were ob- hypothesis that Wolbachia was the cause of this phe- served when wMelPop-infected females blood fed on notype, antibiotic clearance of the wMelPop infection either chicken or mouse blood; however, smaller restored egg development on all hosts to normal levels. fecundity costs of 38Ð45% were observed when On all blood sources, infected and uninfected mos- wMelPop-infected mosquitoes were fed on pig and quitoes fed similarly to repletion, and females as re- sheep blood. As such the mechanistic basis of this vealed by dissection did not retain embryos in their phenotype must rely on factors that vary between ovaries (data not shown), suggesting that general de- individual humans as well as between species. fects in feeding or egg laying behavior are unlikely to The enhanced levels of fecundity and egg viability be contributing factors to this phenomenon. observed when wMelPop-infected females are pro- We also determined that the fecundity of wMelPop- vided with a human bloodmeal in comparison with infected females was reduced relative to antibiotic different animalsÕ blood is intriguing given the natural treated controls when fed human blood, and varied tendency of this mosquito species to blood feed pre- signiÞcantly between human hosts. These results con- dominantly on humans in the Þeld (Scott et al. 1993b, trast with earlier data obtained in the generations Ponlawat and Harrington 2005). The nutritional dif- immediately proceeding establishment of wMelPop in ferences between human and other blood sources that Ae. aegypti, where nonsigniÞcant fecundity costs were promote anthropophilic behavior are currently un- observed when PGYP1 females fed on human blood known (Harrington et al. 2001). Evidence indicates (McMeniman et al. 2009). This discrepancy may pos- however that human blood may potentially be me- sibly reßect 1) an increase in the virulence and asso- tabolized with greater energy efÞciency than other ciated Þtness costs of the wMelPop infection over the types of blood in Ae. aegypti, leading to an accumu- period following transinfection and/or 2) temporal lation of energy reserves (Briegel 1990, Harrington et 82 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 48, no. 1 al. 2001). In this study, variation in the effects of In D. melanogaster, genomic evidence suggests that human and other blood sources suggests that potential Wolbachia obtains much of its energy through metab- interactions between these bacteria and biochemical olism of amino acids (Wu et al. 2004), including thre- differences in host blood may contribute to disruption onineÑone amino acid required for activation of Ae. of egg development. aegypti Vg expression (Attardo et al. 2006). Interest- A decline in egg development after blood feeding ingly, threonine occurs at higher levels in human on mice was not observed in a previous study (Xi et blood relative to guinea pig blood (Chang and Judson al. 2005), where the avirulent wAlbB strain of Wol- 1977)Ña suboptimal blood source for egg production bachia from the mosquito Aedes albopictus (Skuse) in wMelPop-infected Ae. aegypti in this study. As such, was introduced into Ae. aegypti. Likewise, no inter- one mechanistic hypothesis may be that competition actions between any of the blood sources used in this between wMelPop and Ae. aegypti for threonine leads study and egg development have been described for to a decrease in Vg expression, and a subsequent in- other natural, or artiÞcially-generated Wolbachia inhibition of egg development on certain blood sources fections of Culex or Aedes mosquitoes (Dobson et al. low in threonine. In line with a potential interaction 2002, Rasgon and Scott 2003, Xi et al. 2006). Genotypic between wMelPop and mosquito vitellogenesis, the differences between wMelPop and other Wolbachia avirulent Wolbachia strain wMel has recently been strains may thus be responsible for this phenotype in shown to induce small decreases in yolk protein gene Ae. aegypti. expression in previtellogenic ovaries of D. melano- From a mechanistic viewpoint the blood source gaster (Sun and Cline 2009). effect may reßect differences between the metabolic Another well-documented difference in amino acid response of wMelPop relative to other avirulent Wol- levels between the blood sources used in this study is bachia strains to blood feeding. Alternatively, it could the lower isoleucine content of human blood relative relate to the tissue-speciÞc density of wMelPop where to higher levels in mouse, guinea pig, and chicken infection and associated metabolic costs above a phe- blood (Harrington et al. 2001). Previous studies have notypic threshold interferes with the normal process demonstrated that these higher isoleucine levels con- of egg development. The latter of these hypotheses is tribute to the increased fecundity of Ae. aegypti on likely because in Drosophila species, wMelPop exhib- some hosts relative to human blood (Greenberg 1951, its accelerated growth kinetics and increased densities Spielman and Wong 1974, Chang and Judson 1977, in certain tissues relative to infection with avirulent Chang and Judson 1979). However, we observed an Wolbachia strains (Min and Benzer 1997, McGraw et inhibition of egg development when wMelPop-in- al. 2002, McMeniman et al. 2008). fected females fed on blood sources high in isoleucine Egg maturation in Ae. aegypti is a nutrient limited (i.e., mouse, guinea pig, and chicken blood), whereas process that is controlled by a regulated endocrine egg development proceeded normally with low iso- cascade involving several mosquito tissues, including leucine human blood. The potential for an interaction the midgut, hemolymph, brain, ovaries, and fat body between isoleucine levels and wMelPop is not clear (Attardo et al. 2005), all tissues that Wolbachia infect and requires further investigation, because closely re- in other insects (Min and Benzer 1997, Dobson et al. lated Wolbachia strains such as wMel possess biosyn- 1999). Only after a female mosquito takes a bloodmeal thetic pathways for this amino acid (Wu et al. 2004). are the major events of egg maturation activated, in- Alternatively, inhibition of egg development may cluding the accumulation of egg yolk proteins, lipids, be due to reduction of mosquito lipid reserves by and iron by developing oocytes (Raikhel and Dhadi- wMelPop, which are needed for developing oocytes alla 1992, Ziegler and Ibrahim 2001, Zhou et al. 2007). (Ziegler and Ibrahim 2001). It has been hypothesized Both sufÞcient nutritional reserves from larval devel- that Wolbachia may acquire cholesterol for synthesis opment, and absorption of nutrients from the blood- of Lipid A, the major lipid component of the ␣-pro- meal are required for egg development to proceed teobacterial cell wall, by catabolizing host lipid mol- normally (Attardo et al. 2005). Inhibition of egg de- ecules (Lin and Rikihisa 2003, Brownlie and OÕNeill velopment may potentially be the result of competi- 2006). If the cholesterol content of blood sources were tion between Wolbachia and Ae. aegypti for nutrients below a threshold value needed to replenish mosquito that are needed for initiation of mosquito oogenesis or lipid stores or supplement egg development fully dur- the development of oocytes. ing wMelPop infection, a reduction in fecundity and In Ae. aegypti, a key stage of egg development is egg viability could result. A nutritional basis for this vitellogenesis, the tissue-speciÞc expression, synthe- hypothesis is possible, as the range in cholesterol consis, and secretion of the egg yolk protein precursors, tent of human blood is much higher than that of other such as vitellogenin (Vg), by the mosquito fat body animals (Mitruka and Rawnsley 1977). (Attardo et al. 2005). Activation of vitellogenesis is Analogous reductions in fecundity and egg viability dependent on both the presence of the steroid hor- to the wMelPop blood source phenotype have been mone 20-hydroxyecdysone, and amino acids derived observed in studies that have used RNA interference from the digested bloodmeal (Hansen et al. 2004). The to knockdown expression of genes implicated or sus- presence of several speciÞc amino acids is required for pected to function in the nutritional regulation of egg activation of vitellogenesis, with removal of these development in Ae. aegypti. These include genetic leading to a decrease in Vg gene expression (Attardo silencing of 1) the target of rapamycin signaling path- et al. 2006). way required for the nutrient sensitive-activation of January 2011 MCMENIMAN ET AL.: Wolbachia AND Ae. aegypti EGG DEVELOPMENT 83 vitellogenesis in response to amino acids derived from Braks, M. A., S. A. Juliano, and L. P. Lounibos. 2006. Supe- the bloodmeal (Hansen et al. 2004); and 2) the sterol rior reproductive success on human blood without sugar carrier protein AeSCP-2, which is thought to be in- is not limited to highly anthropophilic mosquito species. volved in the uptake of cholesterol and other lipids Med. Vet. Entomol. 20: 53Ð59. across the midgut and fat body for mobilization to Briegel, H. 1990. Metabolic relationship between female developing oocytes (Blitzer et al. 2005). A potential body size, reserves, and fecundity of Aedes aegypti. J. In- interaction between wMelPop infection and amino sect Physiol. 36: 165Ð172. Brownlie, J. C., and S. L. O’Neill. 2006. Wolbachia genom- acid or lipid metabolism in Ae. aegypti therefore war- ics: accelerating our understanding of a pervasive sym- rants further investigation. biosis. In K. Bourtzis and T. A. Miller [eds.], Insect sym- In addition, the recently observed dengue virus biosis, 2nd ed. Taylor & Francis, Boca Raton, FL. resistance phenotype of wMelPop-infected mosqui- Brownlie, J. C., B. N. Cass, M. Riegler, J. J. Witsenburg, I. toes (Moreira et al. 2009) as well as antiviral effects Iturbe-Ormaetxe, E. A. McGraw, and S. L. O’Neill. 2009. observed in Drosophila species carrying avirulent Evidence for metabolic provisioning by a common in- Wolbachia strains, such as wMel and its close relatives vertebrate endosymbiont, Wolbachia pipientis, during pe- (Hedges et al. 2008, Teixeira et al. 2008, Osborne et al. riods of nutritional stress. PLoS Pathog. 5: e1000368. 2009), also may be potentially mediated by competi- Chang, Y. H., and C. L. Judson. 1977. The role of isoleucine tion for limited nutrient resources. As such, it is tempt- in differential egg production by the mosquito Aedes ing to speculate that the fecundity phenotype ob- aegypti Linnaeus (Diptera: Culicidae) following feeding served in this study may be mechanistically linked to on human or guinea pig blood. Comp. Biochem. Physiol. 57A: 23Ð28. viral interference effects of these Wolbachia strains. Chang, Y. H., and C. L. Judson. 1979. Amino acid compo- The identiÞcation of metabolites that differ be- sition of human and guinea pig blood proteins, and ovar- tween host blood sources, and their interaction with ian proteins of the yellow fever mosquito Aedes aegypti; wMelPop infection in Ae. aegypti is needed to char- and their effects on the mosquito egg production Comp. acterize the mechanistic basis of this novel phenotype. Biochem. Physiol. 62A: 753Ð755. We suggest that wMelPop infection may have inad- Cook, P. E., C. J. McMeniman, and S. L. O’Neill. 2008. Mod- vertently unmasked an intrinsic nutritional beneÞt to ifying insect population age structure to control vector- Ae. aegypti feeding on human blood that may form the borne disease. Adv. Exp. Med. Biol. 627: 126Ð140. basis for its strong anthropophilic behavior. Additional Costero, A., J. D. Edman, G. G. Clark, and T. W. Scott. 1998. study of this system may provide new insight into the Life table study of Aedes aegypti (Diptera: Culicidae) in metabolic interplay between Wolbachia and insects Puerto Rico fed only human blood versus blood plus and the nutritional basis of anthropophily. sugar. J. Med. Entomol. 35: 809Ð813. Dobson, S. L., E. J. Marsland, and W. Rattanadechakul. 2002. Mutualistic Wolbachia infection in Aedes albopictus: accelerating cytoplasmic drive. Genetics 160: 1087Ð1094. Acknowledgments Dobson, S. L., K. Bourtzis, H. R. Braig, B. F. Jones, W. Zhou, F. Rousset, and S. L. O’Neill. 1999. Wolbachia infections We thank Kay Marshall, Leon Hugo, and Peter Ryan (all are distributed throughout insect somatic and germ line Queensland Institute of Medical Research) for technical tissues. Insect Biochem. Mol. Biol. 29: 153Ð160. assistance and Elizabeth McGraw, Jeremy Brownlie, Peter Greenberg, J. 1951. 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