Expression of trypsin modulating oostatic factor (TMOF) in an entomopathogenic fungus increases its virulence towards An...

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Title:
Expression of trypsin modulating oostatic factor (TMOF) in an entomopathogenic fungus increases its virulence towards Anopheles gambiae and reduces fecundity in the target mosquito
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Mixed Material
Language:
English
Creator:
Kamareddine, Layla
Fan, Yanhua
Osta, Mike A.
Keyhani, Nemat O.
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BioMed Central (Parasites & Vectors)
Publication Date:

Notes

Abstract:
Background: Adult and larval mosquitoes regulate food digestion in their gut with trypsin modulating oostatic factor (TMOF), a decapeptide hormone synthesized by the ovaries and the neuroendocrine system. TMOF is currently being developed as a mosquitocide, however, delivery of the peptide to the mosquito remains a significant challenge. Entomopathogenic fungi offer a means for targeting mosquitoes with TMOF. Findings: The efficacy of wild type and transgenic Beauveria bassiana strains expressing Aedes aegypti TMOF (Bb-Aa1) were evaluated against larvae and sugar- and blood-fed adult Anopheles gambiae mosquitoes using insect bioassays. Bb-Aa1 displayed increased virulence against larvae, and sugar and blood fed adult A. gambiae when compared to the wild type parent strain. Median lethal dose (LD50) values decreased by ~20% for larvae, and ~40% for both sugar and blood-fed mosquitoes using Bb-Aa1 relative to the wild type parent. Median lethal time (LT50) values were lower for blood-fed compared to sugar-fed mosquitoes in infections with both wild type and Bb-Aa1. However, infection using Bb-Aa1 resulted in 15% to 25% reduction in LT50 values for sugar- and blood fed mosquitoes, and ~27% for larvae, respectively, relative to the wild type parent. In addition, infection with Bb-Aa1 resulted in a dramatic reduction in fecundity of the target mosquitoes. Conclusions: B. bassiana expressing Ae. aegypti TMOF exhibited increased virulence against A. gambiae compared to the wild type strain. These data expand the range and utility of entomopathogenic fungi expressing mosquito-specific molecules to improve their biological control activities against mosquito vectors of disease. Keywords: Trypsin modulating oostatic factor, Biological control, Beauveria bassiana, Anopheles gambiae
General Note:
Publication of this article was funded in part by the University of Florida Open-Access publishing Fund. In addition, requestors receiving funding through the UFOAP project are expected to submit a post-review, final draft of the article to UF's institutional repository, IR@UF, (www.uflib.ufl.edu/UFir) at the time of funding. The institutuional Repository at the University of Florida community, with research, news, outreach, and educational materials.
General Note:
Kamareddine et al. Parasites & Vectors 2013, 6:22 http://www.parasitesandvectors.com/content/6/1/22; Pages 1-5
General Note:
doi:10.1186/1756-3305-6-22 Cite this article as: Kamareddine et al.: Expression of trypsin modulating oostatic factor (TMOF) in an entomopathogenic fungus increases its virulence towards Anopheles gambiae and reduces fecundity in the target mosquito. Parasites & Vectors 2013 6:22.

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p Expression of trypsin modulating oostatic factor (TMOF) in an entomopathogenic fungus increases its virulence towards it Anopheles gambiae and reduces fecundity in the target mosquito
aug
au id A1 snm Kamareddinefnm Laylainsr iid I1 email lyk06@aub.edu.lb
A2 FanYanhuaI2 fanyanhua@gmail.com
A3 ca yes Ostami AMikemo07@aub.edu.lb
A4 KeyhaniONematkeyhani@ufl.edu
insg
ins Department of Biology, American University of Beirut, Bliss Street, Beirut, 11072020, Lebanon
Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, 32611, USA
source Parasites & Vectors
issn 1756-3305
pubdate 2013
volume 6
issue 1
fpage 22
url http://www.parasitesandvectors.com/content/6/1/22
xrefbib pubidlist pubid idtype doi 10.1186/1756-3305-6-22pmpid 23336669
history rec date day 12month 9year 2012acc 1412013pub 2112013
cpyrt 2013collab Kamareddine et al.; licensee BioMed Central Ltd.note This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
kwdg
kwd Trypsin modulating oostatic factor
Biological control
Beauveria bassiana
Anopheles gambiae
abs
sec
st
Abstract
Background
Adult and larval mosquitoes regulate food digestion in their gut with trypsin modulating oostatic factor (TMOF), a decapeptide hormone synthesized by the ovaries and the neuroendocrine system. TMOF is currently being developed as a mosquitocide, however, delivery of the peptide to the mosquito remains a significant challenge. Entomopathogenic fungi offer a means for targeting mosquitoes with TMOF.
Findings
The efficacy of wild type and transgenic Beauveria bassiana strains expressing Aedes aegypti TMOF (Bb-Aa1) were evaluated against larvae and sugar- and blood-fed adult Anopheles gambiae mosquitoes using insect bioassays. Bb-Aa1 displayed increased virulence against larvae, and sugar and blood fed adult A. gambiae when compared to the wild type parent strain. Median lethal dose (LDsub 50) values decreased by ~20% for larvae, and ~40% for both sugar and blood-fed mosquitoes using Bb-Aa1 relative to the wild type parent. Median lethal time (LT50) values were lower for blood-fed compared to sugar-fed mosquitoes in infections with both wild type and Bb-Aa1. However, infection using Bb-Aa1 resulted in 15% to 25% reduction in LT50 values for sugar- and blood fed mosquitoes, and ~27% for larvae, respectively, relative to the wild type parent. In addition, infection with Bb-Aa1 resulted in a dramatic reduction in fecundity of the target mosquitoes.
Conclusions
B. bassiana expressing Ae. aegypti TMOF exhibited increased virulence against A. gambiae compared to the wild type strain. These data expand the range and utility of entomopathogenic fungi expressing mosquito-specific molecules to improve their biological control activities against mosquito vectors of disease.
bdy
Findings
Background
Mosquito vectors transmit numerous diseases to humans and animals, causing illness and death that result in huge socio-economical burdens, especially in endemic countries. Control of mosquito vector populations has been almost exclusively based on the use of insecticidal chemicals; however, the strong dependence on insecticides for mosquito control worldwide and the use of such chemicals in agriculture has led to the physiological resistance of important mosquito vectors in recent years abbrgrp
abbr bid B1 1
B2 2
B3 3
.Entomopathogenic fungi, such as Metarhizium anisopliae and Beauveria bassiana, both EPA approved biological control agents (http://www.epa.gov/pesticides/), offer an environmentally friendly alternative to chemical insecticides, are virulent to mosquitoes, and have been considered as possible candidates for reducing disease transmission by insect vectors
B4 4
B5 5
B6 6
. Despite their potential, widespread use of these fungi remains limited, due to a number of factors that include slow killing speed, low resistance to abiotic stress, issues of spore viability, and the amount of material (spores) needed for effective control. Nevertheless, there is still significant interest in using these biological control agents due to their effectiveness against insecticide resistant mosquito species
B7 7
and advances in the development of tools and lures for their delivery to mosquitoes
B8 8
.Several attempts at increasing the insecticidal effectiveness of entomopathogenic fungi have been based on the development of recombinant DNA techniques. In this context, transgenic entomopathogenic fungi were developed that express potent insect-specific neurotoxins that kill the infected host
B9 9
, or enzymes that enhance fungal resistance to adverse conditions such as UV
B10 10
, ultimately increasing spore viability in nature. In addition to increasing their insecticidal potential, entomopathogenic fungal strains can also be manipulated to target the pathogen, e.g. Plasmodium itself within its mosquito vector
B11 11
.Recently, the idea of using targeted host-specific molecules to subvert normal insect development has been proposed
B12 12
B13 13
. Insects synthesize hormones and neuropeptides that have been exploited as targets for pest control. Examples of these include diuretic hormones and trypsin-modulating oostatic factors (TMOFs) that are found in insects including mosquitoes and flies. Administration of diuretic hormone increased secretion of fluids from the malphigian tubules leading to water loss and death of the insect. The Ae. aegypti TMOF (Aea-TMOF) was shown to circulate in the hemolymph, bind to gut receptors and inhibit trypsin biosynthesis by exerting a translational control on trypsin mRNA
B14 14
. TMOF is resistant to proteolysis in the gut and easily traverses the gut epithelial cells into the hemolymph in adults and larvae, hence, it can be fed to mosquitoes resulting in inhibition of food digestion, anorexia, and is ultimately lethal to the insect
B15 15
B16 16
B17 17
B18 18
. In this report, we measured the virulence of a B. bassiana strain expressing Aea-TMOF to adult (sugar and blood-fed) and larval stages of Anopheles gambiae, the major malaria vector in Africa. The effect of this strain on mosquito fecundity was also determined.
Methods
Anopheles gambiae G3 strain was reared as previously described
B19 19
. Mosquito eggs were treated routinely with 1% Virkon for 4–5 minutes before floating them, to avoid spread of opportunistic infections in the colony. Wild type and Aea-TMOF expressing (Bb-Aa1) B. bassiana strains
B20 20
were cultured at room temperature on potato dextrose agar (PDA) plates for a period of three to four weeks to allow growth of aerial conidia (spores). To collect spores, the surfaces of two B. bassiana PDA plates were scraped using a sterile cell scraper in the presence of sterile ddH2O and the extract was filtered over a glass wool packed column. The flow through material containing spores was centrifuged at 4500 rpm for 10 min and the pellet was washed twice with ddH2O. The pellet was resuspended in ddH2O containing 0.05% Tween 80, spores were counted with a Neubauer hemocytometer and adjusted to the appropriate concentrations for mosquito infections.Adult mosquito bioassays were performed by spraying batches of 45 females (2-days old) each, with spore suspensions of Bb-Aa1 or the wild type (control) strain and mortality was scored on a daily basis. Briefly, adult mosquitoes were anaesthetized on ice, transferred onto a Whatman paper in a Petri dish, sprayed with the appropriate spore suspension using glass atomizers and then returned back to paper cups. Larval bioassays were conducted in plastic trays (8.5 × 13 × 5.5cm) each containing 40 three-days old larvae in a total of 110 ml water. Larvae were infected by applying different concentrations of conidia (1.1 × 10sup 9, 5.5 × 108, 2 × 108 or 1 × 108 conidia in a 50 μl olive oil suspension) to the surface of the assay chamber mixed to achieve homogenous surface spreading. The Kaplan-Meier survival test was used to calculate the percent mortality over the indicated time scale. Statistical significance of the observed differences was calculated by the Log-Rank test. Differences were considered to be significant if P < 0.05. Median lethal time (LT50) values for both B. bassiana strains were calculated from survival curves of adult mosquitoes (both sugar- and blood fed) and larvae infected with 1 × 108 and 5 × 106 spores/ml, respectively, using regression analysis. Statistical analysis of LT50 values was performed using the Student’s T-test. Median lethal dose (LD50) values were determined using concentrations ranging from 1 × 107 to 2 × 108 spores/ml in adult mosquitoes and 9 × 105 to 1 × 107 spores/ml in larvae using the Probit analysis. All experiments were repeated at least three times using different batches of mosquitoes and spores.To examine the effect of fungal infection on mosquito fecundity, A. gambiae females sprayed with a suspension of 1 × 108 spores/ml of wild type or Bb-Aa1 strain, were given a blood meal 24 h after fungal infection. Blood fed females were placed individually into paper cups and eggs were counted 48 h after blood feeding. Statistical analysis was performed using the Mann Whitney test.
Results and discussion
B. bassiana strain Bb-Aa1 expresses Aea-TMOF as a fusion protein with a 28 amino acid signal peptide derived from the B. bassiana chitinase gene to drive the extracellular secretion of the hormone
20
. Sugar and blood-fed adult, female A. gambiae as well as larvae were exposed to spores (conidia) of strain Bb-Aa1 or the wild type parent to determine the effect of Aea-TMOF expression on virulence. Bb-Aa1 was more potent than its wild type parent against both sugar and blood-fed adults (Figure figr fid F1 1A and 1B), causing 40% reduction in LD50 values (50% mortality) in both groups compared to the wild type control (Table tblr tid T1 1). However, LD50 values were similar between sugar and blood-fed mosquitoes infected with the same fungal strain, regardless of its type (Bb-Aa1 or wild type). Infection with Bb-Aa1 also induced a 15 and 25% reduction in the mean survival times (LT50 values) of sugar and blood-fed mosquitoes, respectively, compared to the wild type strain. LT50 values were also lower for blood- compared to sugar fed mosquitoes infected with the same strain, regardless its type. The fact that infections with the same strain, irrespective of its type, resulted in similar LD50 values between sugar and blood-fed mosquitoes, but lowered consistently the LT50 values for the blood- compared to sugar fed group, suggests that the blood meal itself does not seem to affect the virulence of a particular B. bassiana strain but rather mosquito tolerance to infection. Our data are in contrast to Myone et al. (
B21 21
), who reported that blood-fed mosquitoes tended to be similar to or displayed greater median survival times than their unfed cohorts. There are several possible explanations for this discrepancy that can include the assay conditions (Myone et al. used 2 x 1010 conidia/m2 formulated in an oil suspension and sprayed onto sheets) and strain variation (whether fungal or mosquito), and this issue likely deserves greater attention.
fig Figure 1caption Expression of Aea-TMOF increases Beauveria bassiana virulence to Anopheles gambiae mosquitoestext
b Expression of Aea-TMOF increases Beauveria bassiana virulence to Anopheles gambiae mosquitoes. (A) Sugar fed and (B) blood fed adult female A. gambiae mosquitoes were challenged with Bb-Aa1 strain expressing Aea-TMOF or the wild type parent, by spraying with a suspension of 1x108 spores/ml and their survival was scored daily over the indicated period. (C) Three day old larvae were challenged by applying 5.5 x 108 spores (suspended in 50 μl oil) to the water surface and their survival was scored daily over the indicated period as described in the Methods section. Graphs represent percent survival as calculated by the Kaplan-Meier method for one representative experiment of each group. Statistical significance was calculated by the log rank test and survival curves were considered significantly different if P<0.05. (D) Six days after B. bassiana infection, larvae showed significant growth retardation compared to non-infected controls. Wild type B. bassiana (Bb-wt); TMOF-expressing strain (Bb-Aa1). Data shown are representative of at least three independent biological replicates using different batches of mosquitoes and conidia.
graphic file 1756-3305-6-22-1
table
Table 1
LD50 and LT50 values of wild type and
Aea
-TMOF expressing
Bb
-Aa1 strain against
Anopheles gambiae
tgroup align left cols 4
colspec colname c1 colnum 1 colwidth 1*
c2 2
c3 3
c4
thead valign top
row rowsep
entry nameend namest
LD50 and LT50 values of wild-type and TMOF expressing
B. bassiana
infections in
Anopheles gambiae
Strain
Mosquito stage
LD50 (conidia/ml)
LT50 (h)
tfoot
a,c LD50 calculated from the 76 h time point.
e LD50 calculated from the 48 h time point using the Probit analysis.
b,d Bioassays performed by spraying mosquitoes with a concentration of 1x108 spores/ml.
f Larval bioassays performed using a concentration of 5x106 spores/ml.Statistical analysis was performed used the Student’s T-test and values were considered significant if P<0.05.
tbody
Wild-type B. bassiana (sf)
Adult
18.34 ± 2.86 x 107a
133.26 ± 12.79b
Bb: spAeaTMOF (sf)
Adult
11.08 ± 2.79 x 107a (P=0.01)
113.23 ± 17.56b (P=0.009)
Wild-type B. bassiana (bf)
Adult
18.00 ± 2.01 x 107c
115.04 ± 11.26d
Bb: spAeaTMOF (bf)
Adult
11.31 ± 0.01 x 107c (P= 0.04)
86.48 ± 4.68d (P=0.01)
Wild-type B. bassiana
Larvae
14.07 ± 1.24 x 107e
116.31 ± 13.69f
Bb: spAeaTMOF
Larvae
11.32 ± 0.77 x 107e (P=0.01)
85.05 ± 14.66f (P=0.001)
A. gambiae larvae infected with Bb-Aa1 exhibited greater compromised growth and survival as compared to those infected with the wild type strain (Figure 1C). Bb-Aa1 infection of larvae resulted in 20 and 27% reductions in LD50 and LT50 values, respectively, compared to the wild type strain (Table 1). Six days after B. bassiana infection, larvae exhibited significant growth retardation evidenced by their abnormally small size compared to non-infected controls; however, the phenotype was more severe in Bb-Aa1 infected larvae compared to those infected with the wild type strain (Figure 1D).Egg laying was significantly affected by fungal infection. Infection of A. gambiae mosquitoes with the wild-type B. bassiana strain resulted in a significant reduction (~16%) in fecundity compared to non-infected controls (Figure F2 2). However, expression of Aea-TMOF resulted in a dramatic reduction (~60%) in fecundity compared to controls (Figure 2). These data suggest that strain Bb-Aa1 also has the potential to reduce the size of A. gambiae mosquito populations by severely compromising fecundity.
Figure 2Aea-TMOF reduces fecundity of Anopheles gambiae mosquitoes
Aea-TMOF reduces fecundity of Anopheles gambiae mosquitoes. A. gambiae females sprayed with a suspension of 1 × 108 spores/ml of wild type or Bb-Aa1 strain, were given a blood meal 24 h after fungal infection. Non-infected, blood-fed mosquitoes were used as controls. Each circle on the graph represents the number of eggs per mosquito. Medians are indicated by black lines and distributions were compared using the Kolmogorov-Smirnov test. N, number of mosquitoes in each group.
1756-3305-6-22-2
Aea-TMOF does not induce vertebrate toxicity
B22 22
and has passed EPA approval (http://www.epa.gov/pesticides/). Laboratory bioassays showed that the hormone is effective against all the major species of mosquitoes including anopheline
17
. Data from this as well as a previous study
20
indicate that expressing Aea-TMOF in entomopathogenic fungi is a promising approach for the delivery of the hormone to mosquitoes. Entomopathogenic fungi strongly affect mosquito survival
4
B23 23
B24 24
and may constitute a reliable measure for mosquito control when used as part of an integrated vector management plan. A field trial in a malaria endemic village in Tanzania, using experimental huts that simulate local houses, revealed that certain applications of fungal-treated surfaces lead to the infection of approximately 70% of mosquitoes entering the hut, which according to their model, is expected to reduce malaria transmission by 75-80%
B25 25
. Of particular relevance to the data presented here, their model predicts that maintaining a high reduction in malaria transmission rates at lower levels of infected mosquitoes necessitates an increase in fungal virulence
25
. Hence, strains like Bb-Aa1 with improved virulence over standard parent strains are expected to strongly reduce malaria transmission even when the prevalence of fungus-infected mosquitoes is moderate.Fungal strains with a narrower host range and coupled to specific TMOF peptides can also be constructed to maximize specificity and minimize non-target effects. TMOF is a physiologically important host hormone that must be expressed during the life-stages of adults and larvae to control digestion and growth
B26 26
, thus, the mechanisms for resistance to occur can be expected to be life threatening to the mosquito. Despite the fact that TMOF expression increases B. bassiana virulence to A. gambiae, LT50 values of Bb-Aa1 infected mosquitoes suggest that death is not quick enough to completely inhibit females from going through their gonotrophic cycles, even if spores were captured a couple of days before a blood feed. It has been proposed that insecticides exhibiting slow death rates that favor some reproductive success in female mosquitoes can suppress or at least reduce the emergence of resistance
5
. Further, since fungal pathogenesis is a multi-factorial process, where TMOF is not required, but acts to augment virulence, the likelihood of resistance is decreased due to the range of host processes targeted by the fungus, which would have to be overcome. Finally, expression of TMOF does not appear to increase the general virulence of B. bassiana to other insects, i.e. Lepidoptera, indicating target specificity
20
. Ideally, TMOF expressing fungi should be incorporated as part of integrated pest management (IPM) programs that do not rely on a single approach for insect control but which utilize compatible and synergistic approaches.
Conclusions
Our data show that expression of (Aea)-TMOF in a mycoinsecticide can increase its effectiveness against two important mosquito vectors, Ae. aegypti and A. gambiae. Both survival times and the median lethal dose were lower in the Aea-TMOF expressing strain as compared to the wild type parent. In addition, target fecundity was dramatically reduced. Additional research is needed to examine issues of persistence, delivery, and even greater targeting of hosts, however, the principle of exploiting critical target host molecules for expression in an insect pathogen, which, during infection would compromise the host, can be expanded to a wide range of applications in the biological control of insects.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
MAO and NOK conceived the study. YF constructed the fungal strains, LK performed the experimental work. All authors were involved in data analysis and interpretation. MAO and NOK drafted the manuscript. All authors read and approved the final version.
bm
ack
Acknowledgements
We thank Hassan Yassine for helping in mosquito rearing and maintenance and Dr. Riyad Sadek for taking the larval photograph. MAO was funded by AUB-URB and The National Center for Scientific Research, Lebanon. NOK was funded by USDA and NSF grants (2010-34135-21095 and IOS-1121392).
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http:purl.orgdctermsabstract
Abstract
Background
Adult and larval mosquitoes regulate food digestion in their gut with trypsin modulating oostatic factor (TMOF), a decapeptide hormone synthesized by the ovaries and the neuroendocrine system. TMOF is currently being developed as a mosquitocide, however, delivery of the peptide to the mosquito remains a significant challenge. Entomopathogenic fungi offer a means for targeting mosquitoes with TMOF.
Findings
The efficacy of wild type and transgenic Beauveria bassiana strains expressing Aedes aegypti TMOF (Bb-Aa1) were evaluated against larvae and sugar- and blood-fed adult Anopheles gambiae mosquitoes using insect bioassays. Bb-Aa1 displayed increased virulence against larvae, and sugar and blood fed adult A. gambiae when compared to the wild type parent strain. Median lethal dose (LD50) values decreased by ~20% for larvae, and ~40% for both sugar and blood-fed mosquitoes using Bb-Aa1 relative to the wild type parent. Median lethal time (LT50) values were lower for blood-fed compared to sugar-fed mosquitoes in infections with both wild type and Bb-Aa1. However, infection using Bb-Aa1 resulted in 15% to 25% reduction in LT50 values for sugar- and blood fed mosquitoes, and ~27% for larvae, respectively, relative to the wild type parent. In addition, infection with Bb-Aa1 resulted in a dramatic reduction in fecundity of the target mosquitoes.
Conclusions
B. bassiana expressing Ae. aegypti TMOF exhibited increased virulence against A. gambiae compared to the wild type strain. These data expand the range and utility of entomopathogenic fungi expressing mosquito-specific molecules to improve their biological control activities against mosquito vectors of disease.
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Kamareddine, Layla
Fan, Yanhua
Osta, Mike A
Keyhani, Nemat O
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SHORTREPORTOpenAccessExpressionoftrypsinmodulatingoostaticfactor (TMOF)inanentomopathogenicfungusincreases itsvirulencetowards Anophelesgambiae and reducesfecundityinthetargetmosquitoLaylaKamareddine1,YanhuaFan2,MikeAOsta1*andNematOKeyhani2*AbstractBackground: Adultandlarvalmosquitoesregulatefooddigestionintheirgutwithtrypsinmodulatingoostatic factor(TMOF),adecapeptidehormonesynthesizedbytheovariesandtheneuroendocrinesystem.TMOFis currentlybeingdevelopedasamosquitocide,however,deliveryofthepeptidetothemosquitoremainsa significantchallenge.EntomopathogenicfungiofferameansfortargetingmosquitoeswithTMOF. Findings: Theefficacyofwildtypeandtransgenic Beauveriabassiana strainsexpressing Aedesaegypti TMOF( Bb -Aa1) wereevaluatedagainstlarvaeandsugar-andblood-fedadult Anophelesgambiae mosquitoesusinginsectbioassays. BbAa1displayedincreasedvirulenceagainstlarvae,andsugarandbloodfedadult A.gambiae whencomparedtothe wildtypeparentstrain.Medianlethaldose(LD50)valuesdecreasedby~20%forlarvae,and~40%forbothsugarand blood-fedmosquitoesusing Bb -Aa1relativetothewildtypeparent.Medianlethaltime(LT50)valueswerelowerfor blood-fedcomparedtosugar-fedmosquitoesininfectionswithbothwildtypeand Bb -Aa1.However,infectionusing Bb -Aa1resultedin15%to25%reductioninLT50valuesforsugar-andbloodfedmosquitoes,and~27%forlarvae, respectively,relativetothewildtypeparent.Inaddition,infectionwith Bb -Aa1resultedinadramaticreductionin fecundityofthetargetmosquitoes. Conclusions: B.bassiana expressing Ae.aegypti TMOFexhibitedincreasedvirulenceagainst A.gambiae comparedto thewildtypestrain.Thesedataexpandtherangeandutilityofentomopathogenicfungiexpressingmosquito-specific moleculestoimprovetheirbiologicalcontrolactivitiesagainstmosquitovectorsofdisease. Keywords: Trypsinmodulatingoostaticfactor,Biologicalcontrol, Beauveriabassiana AnophelesgambiaeFindingsBackgroundMosquitovectorstransmitnumerousdiseasestohumans andanimals,causingillnessanddeaththatresultinhuge socio-economicalburdens,espe ciallyinendemiccountries. Controlofmosquitovectorp opulationshasbeenalmost exclusivelybasedontheuseofinsecticidalchemicals; however,thestrongdependenceoninsecticidesformosquitocontrolworldwideand theuseofsuchchemicalsin agriculturehasledtothephysiologicalresistanceof importantmosquitovectorsinrecentyears[1-3]. Entomopathogenicfungi,suchas Metarhiziumanisopliae and Beauveriabassiana ,bothEPAapprovedbiological controlagents(http://www.epa .gov/pesticides/),offeran environmentallyfriendlyalternativetochemicalinsecticides,arevirulenttomosquitoes,andhavebeenconsidered aspossiblecandidatesforreducingdiseasetransmissionby insectvectors[4-6].Despiteth eirpotential,widespreaduse ofthesefungiremainslimited,duetoanumberoffactors thatincludeslowkillingspeed,lowresistancetoabiotic stress,issuesofsporeviability,andtheamountofmaterial (spores)neededforeffectivecon trol.Nevertheless,thereis stillsignificantinterestinu singthesebiologicalcontrol *Correspondence: mo07@aub.edu.lb ; keyhani@ufl.edu1DepartmentofBiology,AmericanUniversityofBeirut,BlissStreet,Beirut 11072020,Lebanon2DepartmentofMicrobiologyandCellScience,UniversityofFlorida, Gainesville,FL32611,USA 2013Kamareddineetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse, distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.Kamareddine etal.Parasites&Vectors 2013, 6 :22 http://www.parasitesandvectors.com/content/6/1/22

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agentsduetotheireffectivenessagainstinsecticideresistant mosquitospecies[7]andadvancesinthedevelopmentof toolsandluresfortheirdeliverytomosquitoes[8]. Severalattemptsatincreasingtheinsecticidaleffectivenessofentomopathogenicfungihavebeenbasedonthe developmentofrecombinantDNAtechniques.Inthiscontext,transgenicentomopathogenicfungiweredeveloped thatexpresspotentinsect-spec ificneurotoxinsthatkillthe infectedhost[9],orenzymesthatenhancefungalresistance toadverseconditionssuchasUV[10],ultimatelyincreasing sporeviabilityinnature.Ina dditiontoincreasingtheir insecticidalpotential,ento mopathogenicfungalstrains canalsobemanipulatedtotargetthepathogen,e.g. Plasmodium itselfwithinitsmosquitovector[11]. Recently,theideaofusingtargetedhost-specificmoleculestosubvertnormalinsectdevelopmenthasbeen proposed[12,13].Insectssynthesizehormonesandneuropeptidesthathavebeenexploitedastargetsforpestcontrol. Examplesoftheseincludediur etichormonesandtrypsinmodulatingoostaticfactors(TMOFs)thatarefoundin insectsincludingmosquitoesandflies.Administrationof diuretichormoneincreasedsecretionoffluidsfromthe malphigiantubulesleadingtowaterlossanddeathofthe insect.The Ae.aegypti TMOF( AeaTMOF)wasshownto circulateinthehemolymph,bindtogutreceptorsand inhibittrypsinbiosynthesisbyexertingatranslationalcontrolontrypsinmRNA[14].TMOFisresistanttoproteolysisinthegutandeasilytraversesthegutepithelialcellsinto thehemolymphinadultsandlarvae,hence,itcanbefedto mosquitoesresultingininhib itionoffooddigestion,anorexia,andisultimatelylethaltotheinsect[15-18].Inthis report,wemeasuredthevirulenceofa B.bassiana strain expressing Aea -TMOFtoadult(sugarandblood-fed)and larvalstagesof Anophelesgambiae ,themajormalaria vectorinAfrica.Theeffectofthisstrainonmosquito fecunditywasalsodetermined.MethodsAnophelesgambiae G3strainwasrearedaspreviously described[19].Mosquitoeggsweretreatedroutinelywith 1%Virkonfor4 – 5minutesbeforefloatingthem,toavoid spreadofopportunisticinfectionsinthecolony.Wildtype and Aea -TMOFexpressing( Bb -Aa1) B.bassiana strains [20]wereculturedatroomtemperatureonpotato dextroseagar(PDA)platesforaperiodofthreetofour weekstoallowgrowthofaerialconidia(spores).Tocollect spores,thesurfacesoftwo B.bassiana PDAplateswere scrapedusingasterilecellscraperinthepresenceofsterileddH2Oandtheextractwasfilteredoveraglasswool packedcolumn.Theflowthroughmaterialcontaining sporeswascentrifugedat4500rpmfor10minandthe pelletwaswashedtwicewithddH2O.Thepelletwas resuspendedinddH2Ocontaining0.05%Tween80,spores werecountedwithaNeubauerhemocytometerand adjustedtotheappropriateconcentrationsformosquito infections. Adultmosquitobioassayswe reperformedbyspraying batchesof45females(2-daysold)each,withsporesuspensionsof Bb -Aa1orthewildtype(control)strainandmortalitywasscoredonadailybasis.Briefly,adultmosquitoes wereanaesthetizedonice,transferredontoaWhatman paperinaPetridish,sprayedwiththeappropriatespore suspensionusingglassatomizersandthenreturnedbackto papercups.Larvalbioassayswereconductedinplastictrays (8.5135.5cm)eachcontaining40three-daysoldlarvae inatotalof110mlwater.Larvaewereinfectedbyapplying differentconcentrationsofconidia(1.1109,5.5108, 2108or1108conidiaina50 loliveoilsuspension)to thesurfaceoftheassaychambermixedtoachieve homogenoussurfacespreading.TheKaplan-Meiersurvival testwasusedtocalculatethepercentmortalityoverthe indicatedtimescale.Statisticalsignificanceoftheobserved differenceswascalculatedbytheLog-Ranktest.Differences wereconsideredto besignificantif P <0.05.Medianlethal time(LT50)valuesforboth B.bassiana strainswerecalculatedfromsurvivalcurvesofadultmosquitoes(bothsugarandbloodfed)andlarvaeinfectedwith1108and5106spores/ml,respectively,usingre gressionanalysis.Statistical analysisofLT50valueswasperformedusingtheStudent ’ s T-test.Medianlethaldose(LD50)valuesweredetermined usingconcentrations rangingfrom1107to2108spores/mlinadultmosquitoesand9105to1107spores/mlinlarvaeusingtheProbitanalysis.Allexperimentswererepeatedatleastthreetimesusingdifferent batchesofmosquitoesandspores. Toexaminetheeffectoffungalinfectiononmosquito fecundity, A.gambiae femalessprayedwithasuspension of1108spores/mlofwildtypeor Bb -Aa1strain,were givenabloodmeal24hafterfungalinfection.Bloodfed femaleswereplacedindividuallyintopapercupsand eggswerecounted48hafterbloodfeeding.Statistical analysiswasperformedusingtheMannWhitneytest.ResultsanddiscussionB.bassiana strain Bb-Aa1 expresses AeaTMOFasafusion proteinwitha28aminoacids ignalpeptidederivedfrom the B.bassiana chitinasegenetodrivetheextracellular secretionofthehormone[20].Sugarandblood-fedadult, female A.gambiae aswellaslarvaewereexposedtospores (conidia)ofstrain Bb -Aa1orthewildtypeparentto determinetheeffectof Aea -TMOFexpressionon virulence. Bb-Aa1 wasmorepotentthanitswildtype parentagainstbothsugarandblood-fedadults(Figure1A and1B),causing40%reductioninLD50values(50% mortality)inbothgroupscomparedtothewildtype control(Table1).However,LD50valuesweresimilar betweensugarandblood-fedmosquitoesinfectedwith thesamefungalstrain,regardlessofitstype( Bb -Aa1orKamareddine etal.Parasites&Vectors 2013, 6 :22 Page2of5 http://www.parasitesandvectors.com/content/6/1/22

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wildtype).Infectionwith Bb -Aa1alsoinduceda15and 25%reductioninthemeansurvivaltimes(LT50values)of sugarandblood-fedmosquitoes,respectively,compared tothewildtypestrain.LT50valueswerealsolowerfor blood-comparedtosugarfedmosquitoesinfectedwith thesamestrain,regardlessitstype.Thefactthatinfections withthesamestrain,irrespectiveofitstype,resultedin similarLD50valuesbetweensugarandblood-fedmosquitoes,butloweredconsistentlytheLT50valuesforthe blood-comparedtosugarfedgroup,suggeststhatthe bloodmealitselfdoesnotseemtoaffectthevirulenceofa particular B.bassiana strainbutrathermosquitotolerance toinfection.OurdataareincontrasttoMyone etal. ([21]),whoreportedthatblood-fedmosquitoestendedto besimilartoordisplayedgreatermediansurvivaltimes thantheirunfedcohorts.Thereareseveralpossibleexplanationsforthisdiscrepancythatcanincludetheassay conditions(Myone etal. used2x1010conidia/m2formulatedinanoilsuspensionandsprayedontosheets)and strainvariation(whetherfungalormosquito),andthisissue likelydeservesgreaterattention. A.gambiae larvaeinfectedwith Bb -Aa1exhibitedgreater compromisedgrowthandsurvivalascomparedtothose infectedwiththewildtypestrain(Figure1C). Bb -Aa1 infectionoflarvaeresultedin20and27%reductionsin LD50andLT50values,respectively,comparedtothewild typestrain(Table1).Sixdaysafter B.bassiana infection, larvaeexhibitedsignificantgrowthretardationevidenced bytheirabnormallysmallsizecomparedtonon-infected controls;however,thephenotypewasmoreseverein Bb -Aa1infectedlarvaecomparedtothoseinfected withthewildtypestrain(Figure1D). Egglayingwassignificantlyaffectedbyfungalinfection. Infectionof A.gambiae mosquitoeswiththewild-type B. bassiana strainresultedinasignificantreduction(~16%) infecunditycomparedtonon-infectedcontrols(Figure2). However,expressionof AeaTMOFresultedinadramatic reduction(~60%)infecunditycomparedtocontrols Figure1 Expressionof Aea -TMOFincreases Beauveriabassiana virulenceto Anophelesgambiae mosquitoes. ( A )Sugarfedand( B )blood fedadultfemale A.gambiae mosquitoeswerechallengedwith Bb -Aa1strainexpressing Aea -TMOForthewildtypeparent,bysprayingwitha suspensionof1x108spores/mlandtheirsurvivalwasscoreddailyovertheindicatedperiod.( C )Threedayoldlarvaewerechallengedby applying5.5x108spores(suspendedin50 loil)tothewatersurfaceandtheirsurvivalwasscoreddailyovertheindicatedperiodasdescribed intheMethodssection.GraphsrepresentpercentsurvivalascalculatedbytheKaplan-Meiermethodforonerepresentativeexperimentofeach group.Statisticalsignificancewascalculatedbythelogranktestandsurvivalcurveswereconsideredsignificantlydifferentif P <0.05.( D )Sixdays after B.bassiana infection,larvaeshowedsignificantgrowthretardationcomparedtonon-infectedcontrols.Wildtype B.bassiana ( Bb -wt);TMOFexpressingstrain( Bb -Aa1).Datashownarerepresentativeofatleastthreeindependentbiologicalreplicatesusingdifferentbatchesof mosquitoesandconidia. Kamareddine etal.Parasites&Vectors 2013, 6 :22 Page3of5 http://www.parasitesandvectors.com/content/6/1/22

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(Figure2).Thesedatasuggestthatstrain Bb -Aa1alsohas thepotentialtoreducethesizeof A.gambiae mosquito populationsbyseverelycompromisingfecundity. Aea -TMOFdoesnotinducevertebratetoxicity[22] andhaspassedEPAapproval(http://www.epa.gov/ pesticides/).Laboratorybioassaysshowedthatthehormoneiseffectiveagainstallthemajorspeciesofmosquitoesincludinganopheline[17].Datafromthisas wellasapreviousstudy[20]indicatethatexpressing AeaTMOFinentomopathogenicfungiisapromising approachforthedeliveryofthehormonetomosquitoes.Entomopathogenicfungistronglyaffectmosquito survival[4,23,24]andmayco nstituteareliablemeasureformosquitocontrolwhenusedaspartofan integratedvectormanagementplan.Afieldtrialina malariaendemicvillageinTanzania,usingexperimentalhutsthatsimulatelocalhouses,revealedthatcertainapplicationsoffungal-treatedsurfacesleadtothe infectionofapproximately70%ofmosquitoesenteringthehut,whichaccordingtotheirmodel,is expectedtoreducemalariatransmissionby75-80% [25].Ofparticularrelevancetothedatapresentedhere, theirmodelpredictsthatmaintainingahighreductionin malariatransmissionratesatlowerlevelsofinfectedmosquitoesnecessitatesanincreaseinfungalvirulence[25]. Hence,strainslike Bb -Aa1withimprovedvirulence overstandardparentstrain sareexpectedtostrongly reducemalariatransmissionevenwhentheprevalence offungus-infectedmosquitoesismoderate. Fungalstrainswithanarrowerhostrangeand coupledtospecificTMOFpeptidescanalsobeconstructedtomaximizespecificityandminimizenontargeteffects.TMOFisaphysiologicallyimportant hosthormonethatmustbeexpressedduringthelifestagesofadultsandlarvaetocontroldigestionand growth[26],thus,themechanismsforresistanceto occurcanbeexpectedtobelifethreateningtothe mosquito.DespitethefactthatTMOFexpression increases B.bassiana virulenceto A.gambiae ,LT50valuesof Bb -Aa1infectedmosquitoessuggestthat deathisnotquickenoughtocompletelyinhibit femalesfromgoingthroughtheirgonotrophiccycles, evenifsporeswerecapturedacoupleofdaysbefore abloodfeed.Ithasbeenproposedthatinsecticides exhibitingslowdeathratesthatfavorsomereproductivesuccessinfemalemosquitoescansuppressorat leastreducetheemergenceofresistance[5].Further, sincefungalpathogenesisisamulti-factorialprocess, whereTMOFisnotrequired,butactstoaugment virulence,thelikelihoodofresistanceisdecreaseddue totherangeofhostprocessestargetedbythefungus, whichwouldhavetobeovercome.Finally,expression ofTMOFdoesnotappeartoincreasethegeneral virulenceof B.bassiana tootherinsects,i.e.Lepidoptera,indicatingtargetspecificity[20].Ideally,TMOF expressingfungishouldbeincorporatedaspartof integratedpestmanagement(IPM)programsthatdo notrelyonasingleapproachforinsectcontrolbut whichutilizecompatibleandsynergisticapproaches. Figure2 Aea -TMOFreducesfecundityof Anophelesgambiae mosquitoes. A.gambiae femalessprayedwithasuspensionof 1108spores/mlofwildtypeor Bb -Aa1strain,weregivenablood meal24hafterfungalinfection.Non-infected,blood-fedmosquitoes wereusedascontrols.Eachcircleonthegraphrepresentsthe numberofeggspermosquito.Mediansareindicatedbyblacklines anddistributionswerecomparedusingtheKolmogorov-Smirnov test.N,numberofmosquitoesineachgroup. Table1LD50andLT50valuesofwildtypeand Aea -TMOF expressing Bb -Aa1strainagainst AnophelesgambiaeLD50andLT50valuesofwild-typeandTMOFexpressing B. bassiana infectionsin Anophelesgambiae StrainMosquito stage LD50(conidia/ml)LT50(h) Wild-type B. bassiana (sf) Adult18.342.86x107a133.2612.79bBb:spAeaTMOF(sf)Adult11.082.79x107a( P =0.01) 113.2317.56b( P =0.009) Wild-type B. bassiana (bf) Adult18.002.01x107c115.0411.26dBb:spAeaTMOF(bf)Adult11.310.01x107c( P =0.04) 86.484.68d( P =0.01) Wild-type B.bassiana Larvae14.071.24x107e116.3113.69fBb:spAeaTMOFLarvae11.320.77x107e( P =0.01 ) 85.0514.66f( P =0.001)a,cLD50calculatedfromthe76htimepoint.eLD50calculatedfromthe48htimepointusingtheProbitanalysis.b,dBioassaysperformedbysprayingmosquitoeswithaconcentrationof1x108spores/ml.fLarvalbioassaysperformedusingaconcentrationof5x106spores/ml. StatisticalanalysiswasperformedusedtheStudent ’ sT-testandvalueswere consideredsignificantif P <0.05.Kamareddine etal.Parasites&Vectors 2013, 6 :22 Page4of5 http://www.parasitesandvectors.com/content/6/1/22

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ConclusionsOurdatashowthatexpressionof( Aea )-TMOFina mycoinsecticidecanincreaseitseffectivenessagainsttwo importantmosquitovectors, Ae.aegypti and A.gambiae Bothsurvivaltimesandthemedianlethaldosewerelower intheAea-TMOFexpressingstrainascomparedtothe wildtypeparent.Inaddition,targetfecunditywasdramaticallyreduced.Additionalresearchisneededtoexamine issuesofpersistence,delivery,andevengreatertargeting ofhosts,however,theprincipleofexploitingcriticaltarget hostmoleculesforexpressioninaninsectpathogen, which,duringinfectionwouldcompromisethehost,can beexpandedtoawiderangeofapplicationsinthe biologicalcontrolofinsects.Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Authors ’ contributions MAOandNOKconceivedthestudy.YFconstructedthefungalstrains,LK performedtheexperimentalwork.Allauthorswereinvolvedindataanalysis andinterpretation.MAOandNOKdraftedthemanuscript.Allauthorsread andapprovedthefinalversion. Acknowledgements WethankHassanYassineforhelpinginmosquitorearingandmaintenance andDr.RiyadSadekfortakingthelarvalphotograph.MAOwasfundedby AUB-URBandTheNationalCenterforScientificResearch,Lebanon.NOKwas fundedbyUSDAandNSFgrants(2010-34135-21095andIOS-1121392). Received:12September2012Accepted:14January2013 Published:21January2013 References1.KoekemoerLL,SpillingsBL,ChristianRN,LoTC,KaiserML,NortonRA,Oliver SV,ChoiKS,BrookeBD,HuntRH, etal : MultipleInsecticideResistancein Anophelesgambiae(Diptera:Culicidae)fromPointeNoire,Republicof theCongo. VectorBorneZoonoticDis 2011, 11 (8):1193 – 1200. 2.LabbeP,BerthomieuA,BerticatC,AloutH,RaymondM,LenormandT,Weill M: Independentduplicationsoftheacetylcholinesterasegeneconferring insecticideresistanceinthemosquitoCulexpipiens. MolBiolEvol 2007, 24 (4):1056 – 1067. 3.LimaEP,PaivaMH,deAraujoAP,daSilvaEV,daSilvaUM,deOliveiraLN, SantanaAE,BarbosaCN,dePaivaNetoCC,GoulartMO, etal : Insecticide resistanceinAedesaegyptipopulationsfromCeara. 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