Ep

GMBa

b

c

d

e

U

a

ARR1AA

KALMSSL

1

wifoaotmim

d

0h

Acta Tropica 132S (2014) S96–S101

Contents lists available at ScienceDirect

Acta Tropica

jo u r n al homep age: www.elsev ier .com/ locate /ac ta t ropica

ffect of three larval diets on larval development and male sexualerformance of Anopheles gambiae s.s.

ildas A. Yahouédoa, Luc Djogbénoua,∗, Jacques Saïzonoua, Bénoît S. Assogbaa,ichel Makoutodéa, Jeremie R.L. Gillesc, Hamidou Maïgad, Karine Moulined,e,

honna K. Soukoub, Frédéric Simarde

Institut Régional de Santé Publique, Université d’Abomey Calavi, 01BP918 Cotonou, BeninCentre de Recherche Entomologique de Cotonou (CREC), Cotonou, BeninInsect Pest Control Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food, International Atomic Energy Agency, Vienna, AustriaInstitut de Recherche en Science de la Santé/Centre Muraz, BP 545, Bobo-Dioulasso, Burkina FasoInstitut de Recherche pour le Développement (IRD), Maladies Infectieuses et Vecteurs: Ecologie, Génétique, Evolution et Contrôle (MIVEGEC), UMRM1-UM2 - CNRS 5290 – IRD 224, Montpellier, France

r t i c l e i n f o

rticle history:eceived 30 August 2013eceived in revised form7 November 2013ccepted 19 November 2013vailable online 27 November 2013

eywords:nopheles gambiae s.s.arval developmentale fitness

a b s t r a c t

Population replacement/elimination strategies based on mass-release of sterile or otherwise geneticallymodified (male) mosquitoes are being considered in order to expand the malaria vector control arsenalon the way to eradication. A challenge in this context, is to produce male mosquitoes that will be able tocompete and mate with wild females more efficiently than their wild counterparts, i.e. high fitness males.This study explored the effect of three larval food diets developed by the International Atomic EnergyAgency on the overall fitness and mating performance of male Anopheles gambiae s.s. mosquitoes (Kisumustrain). Larval development (pupation and emergence rate, development time) was monitored, and adultwing length and energy reserves at emergence (i.e. lipids, sugars, glycogen and proteins) were measured.Male sexual performance was assessed through an insemination test whereby one male and 10 virginfemales were maintained together in the same cage in order to record the number of inseminated females

exual competitionITarval diets

per 24 h. Our results show that males reared on Diets 2 and 3 performed best during larval development.Males provided with treatment 2.2 had a shorter development time and performed best in inseminationtests. However, these males had the lowest overall lifespan, suggesting a trade-off between longevity andsexual performances which needs to be taken into consideration when planning release. The results fromthis work were discussed in the context of sterile insect techniques or genetic control methods which is

in thnal A

today one of the strategyCopyright © Internatio

. Introduction

Malaria control relies on prompt and effective treatmentith drug combinations containing artemisinin and the use of

nsecticide-treated nets and indoor residual insecticide sprayingor vector control (WHO, 2001). However, today in many partsf the world, the parasites have developed resistance to manyntimalarial drugs as have mosquitoes to the different familiesf insecticides used in public health (WHO, 2001). Currently, vec-or control represents the mainstay for reducing the incidence of

alaria in sub-Saharan regions of Africa, and due to the currentnsecticide resistance problems, it is imperative to find alternative

ethods (Dabiré et al., 2008; Djogbénou et al., 2010), among

∗ Corresponding author at: Institut Régional de Santé Publique/Université’Abomey Calavi, BP: 384 Ouidah & 01 BP 918, Cotonou, Benin. Tel.: +229 21341674.

E-mail address: ldjogbenou22002@yahoo.fr (L. Djogbénou).

001-706X/$ – see front matter. Copyright © International Atomic Energy Agency 2013. Pttp://dx.doi.org/10.1016/j.actatropica.2013.11.014

e overall mosquito control and elimination efforts.tomic Energy Agency 2013. Published by Elsevier B.V. All rights reserved.

which are genetic control and the sterile insect technique (SIT)(Catteruccia et al., 2009; Cohuet et al., 2004; Helinski et al.,2006; Klassen, 2009). The use of laboratory reared sterile malesis under consideration as a novel way of controlling Anophelesvectors of malaria. Such projects may work in isolated popula-tions such as though found on islands. In particular a project isbeing prepared under the auspices of the International AtomicEnergy Agency (IAEA) of the release of irradiated sterile males ofAnopheles arabiensis in the Dongola area of Sudan. The suppres-sion of insect populations using SIT has been used in agriculturefor over fifty years to control pests (Baumhover, 2002; Cacereset al., 2004). Since female mosquitoes only mate once in theirlife sterile males may induce females to lay sterile eggs andso reduce the population. If a sufficient number of females are

mated in such a way the reproductive rate may be reduced toless than one, leading eventually to population elimination. Lab-oratory reared mosquitoes have had to go through a number ofgenetic bottlenecks in order to adapt to life inside a cage. This

ublished by Elsevier B.V. All rights reserved.

Tropi

tut(rtmcmwm

ms(caalat11bieaamafl2thti2ud

ptcrutddfisoieeatmc

2

2

A

G.A. Yahouédo et al. / Acta

ends to reduce their fitness compared to wild males. Programssing SIT in the early 1960s in the USA against An. quadrimacula-us and Aedes aegypti had no significant effect on wild populationsWeidhaas, 1962; Benedict and Robinson, 2003). Similarly, theelease of sterile males An. culicifacies in Pakistan did not producehe desired effect due to the low sexual competitiveness of released

ales (Rathor et al., 1980). Given these failures, it is essential toonduct fundamental laboratory and field studies on the deter-inants of survival and male sexual performance and means byhich related factors of insect biology can be altered and opti-ized.The success of SIT depends largely on the ability of male

osquitoes to live long enough to mate, to efficiently locate theirexual partners and to be at least as competitive as wild malesAlphey et al., 2010). These characteristics are all part of the con-ept of ‘fitness’ associated with reproduction. In mosquitoes, thequatic (larval) phase is paramount. During this stage, larvae feednd accumulate substances relevant for their adult life and particu-arly emergence and first flight. Larval development time, adult size,nd adult longevity are biological parameters that contribute tohe sexual performance of mosquitoes (Ameneshewa and Service,996; Armbruster and Hutchinson, 2002; Briegel, 1990c; Hawley,985; Hogg et al., 1996; Lyimo and Takken, 1993). Reproductiveehavior in An. gambiae is mainly based on the formation of mat-

ng swarms (Charlwood et al., 2003; Diabate et al., 2003; Diabatét al., 2006) which are an aggregation of male mosquitoes that formt dusk and last about 30 min (Charlwood and Jones, 1979; Yuvalnd Bouskila, 1993). For males to enter this mating arena, theyust have sufficient energy resources in the form of lipids, sugar

nd glycogen reserves. Even if mosquitoes feed preferentially onower nectar (their energy source in the field) (Foster and Takken,004), reserves accumulated during the larval stage contribute toheir successful participation in swarming. Indeed, many studiesave shown that several species of insects can directly reallocateheir food reserves stored during the larval phase to other functionsn the adult stage (Boggs and Freeman, 2005; Reim and Teuschl,006; Kooijman, 2000), hence the close relationship between sex-al performance of males and their larval development, includingiet (Ng’habi et al., 2005).

A major concern for entomologists and managers of integratedest management programmes that include SIT is whether or nothe released sterile males compete well with wild males, and suc-essfully mate and inseminate the targeted wild females. For thiseason, mass rearing of males An. gambiae with sufficient sex-al performance is one of the most important components inhese programs. To achieve high phenotypic quality, optimal con-itions for mass-rearing need to be defined which include larvaliets and feeding regimes. In this paper we compared three arti-cial larval diets developed by the IAEA for An. gambiae (Kisumutrain). We evaluated the effects of these diets on larval devel-pment and the phenotypic quality of emerging adult males,ncluding their sexual performance. We measured pupation andmergence rate, larval development time, adult wing size andnergy reserves (i.e. lipids, sugars, glycogen and protein content)t emergence in male mosquitoes. Furthermore, we explored,hrough an insemination test, the effect on male sexual perfor-

ance of larval rearing with each of the three diets at differentoncentrations.

. Materials and methods

.1. Mosquito strain

All experiments were carried out with the Kisumu strain ofn. gambiae s.s. (S molecular form), a laboratory reference strain

ca 132S (2014) S96–S101 S97

susceptible to all insecticides currently used in public health. Exper-iments were performed in the larval insectary (temperature 28 ± 2and humidity around 80%) of Institut Régional de Santé Publiquebased at Ouidah, Benin.

2.2. Larval diets

Three custom diets produced by IAEA (Vienna, Austria) wereused for larval rearing: Diet 1 (Bovine Liver Powder + TunaMeal + Vitamin Mix), Diet 2 (Bovine Liver Powder + Brewer’sYeast + Tuna Meal + Squid liver powder + Vitamin Mix), Diet 3 (KoiFish Food + Vitamin Mix). Two concentrations of each of these dietswere tested (1% and 2%, w/v solutions) and we renamed the treat-ments: 1.1, 1.2, 2.1, 2.2, 3.1, 3.2 corresponding to Diet 1 at 1% dosage,Diet 1 used at 2%, Diet 2 used at 1%, Diet 2 used at 2%, Diet 3 usedat 1% and Diet 3 used at 2%, respectively. Thus we had six treat-ments which were previously prepared and distributed into 1.5 mlEppendorf tubes and stored at −20 ◦C before use, following IAEAguidelines (Puggioli et al., 2013).

2.3. Larval development assays and monitoring

This first experiment was conducted to determine the type oftreatment (diet) and the concentration necessary to optimize larvaldevelopment in An. gambiae s.s. All experiments were performed ona density of one first instar larvae (L1) per ml. For this purpose,eggs matured for one day were collected and put into a plasticbowls containing 1L of deionized water without food. The wholeeggs were from Kisumu adults reared in laboratory. At hatching, 32L1 larvae were counted and carefully transferred into petri dishes(9 cm in diameter) filled with 32 ml of deionized water, labeled andcovered. The petri dishes (3 per treatment) were arranged ran-domly on a shelf covered with a white background to facilitatelarval counting. All experimental treatments were tested simul-taneously. Every day, the different quantities of diet (0.2 or 0.4 mgdiet dilution/larvae/day) were provided to each petri dish by adding640 �l of the 1 or 2% (w:v). Petri dishes disposition on the shelfwas changed every day. Dead larvae were recorded and removed.Once L4 larval were present, dishes were checked every hour forthe presence of pupae. When observed, the pupae were removedand placed in tubes plugged with cotton wool and containing 1 mlof water labeled with the date, treatment, time and number of thedish they pupated from, and left to emerge. Three replicates of eachtreatment were performed (3 petri dishes per treatment), givinga total number of 1152 L1 larval that were monitored (32 L1 × 3replicates × 6 treatments × 2 experiments).

We monitored (i) pupation rate, (ii) emergence rate, and (iii)larval development time. For each treatment and each replicate,the pupation rate was determined from the ratio of total pupaeobtained to the total number of L1 larvae at the beginning of theexperiment and expressed as a percentage for each treatment.

Emergence rate was assessed for each petri dish as the ratio ofemerging adults to the total number of L1 larvae at the beginning ofthe experiment and expressed as a percentage for each treatment.Larval development time was the number of days for each malemosquito provided with each treatment to reach the adult stage.

2.4. Phenotypic quality of emerging male mosquitoes

Upon emergence, male mosquitoes were randomly sampled andtheir wings were dissected and mounted between slides and cover-slips. They were photographed using a binocular microscope (Leica,

Switzerland). Images of wings were then measured using ImageJ1.41.0 software (Wayne Rasband National Institutes of Health,USA) and wing length (WL) was measured from the alula notchto the distal end (Charlwood, 1996). This value cubed (WL3) was

S Tropica 132S (2014) S96–S101

uB

s2kplTfdw(meB

2

wovgmE1t(swemoftatdttep

2

dututt

Fig. 1. Survival curve of L1 larvae according to different concentrations and diets.

TD

M

98 G.A. Yahouédo et al. / Acta

sed as a proxy for mosquito size (Briegel, 1990a; Fernandes andriegel, 2005).

The metabolites present in emerging adult males were mea-ured on single mosquitoes (N = 10 per treatment) maintained for4 h after emergence with access to water only. They were rapidlyilled in liquid nitrogen and stored at −80 ◦C until the assay waserformed. One wing of each male was dissected under a binocu-

ar microscope and its length was measured as described above.otal lipid, protein, glycogen and sugars content was assessedor each single mosquito in each treatment, using previouslyescribed methods (Rivero et al., 2007). Control data was recordedith male reared with standard laboratory food at a same doses

TetraMikromin, Tetra Werke, Germany). The energy reserves ofales were adjusted to their body size and the results were

xpressed in Joule per cubic millimeter (J/mm3) (Fernandes andriegel, 2005).

.5. Insemination test

The male mosquitoes from the previous rearing experimentere used for a test of insemination ability. Single male (2-day

ld) reared with different treatments were put together with 10irgin females (2-day old) in a cage (30 cm × 30 cm × 30 cm). Vir-in females used were caged under the same conditions as theales above. Mosquitoes were provided with a 10% sugar solution.

very 24 h, these females were removed and replaced with others0 virgin females (2-day old) from the same mosquito colony. Theest was conducted for 15 consecutive days with the same malesbecause after 15 days there is no more effective insemination: per-onal observation). Females were killed and their spermathecaeere dissected in PBS under a binocular microscope. Spermath-

cae were broken, the release of any sperm observed under 40×agnification, and females were scored as positive (inseminated)

r negative (no sperm present in spermathecae). The number ofemales inseminated by each male mosquito out of the ten washen recorded. Finally, a score was assigned to each mosquito: theverage number of females inseminated per 24 h during 15 days ofesting. Furthermore we plotted the sum of females inseminateduring the male lifespans. Five replicates were performed for eachreatment group. The day on which insemination took place andhe death day of each male involved in this study was recorded tovaluate the parameters “period” (day on which insemination tooklace) and “lifespan” of the adult male.

.6. Statistical analysis

All data were entered into Excel 2010. For comparison of larvalevelopment parameters, a logistic regression (quasibinomial) wassed to estimate the coefficients and odds-ratio associated with

he glm function of the R software. The Kaplan–Meier method wassed to estimate the duration of larval development. In addition,he relative chances (probability to obtain an adult mosquito in onereatment compared to another) at each time of their development

able 1evelopment parameters (mean ± SE) and wing length (mm) of adult males of Anopheles

Treatments Parameters

N. pupae N. adult

1.1 21.67 ± 0.36ab 18.67 ± 0.30a1.2 25.33 ± 0.43ab 20.5 ± 0.64a2.1 23.5 ± 0.31ab 18.33 ± 0.12a2.2 25.5 ± 0.28ab 23.5 ± 0.37 c3.1 22.17 ± 0.45a 14.33 ± 0.72b3.2 28.33 ± 0.19cb 25 ± 0.26d

eans (based on three replicates) within a row followed by the same letter are not signifi

The intersection of the different curves by the dotted line show the necessary devel-opment time for 50% of larvae to reach the adult stage. “Final status” means reachingadult stage.

was determined through a model of proportional hazard regressionbased on the function COXPH with the Breslow’s approximation.The wing length of newly emerged males and their energy reserveswere compared using a linear regression (ANOVA, R software). Inthe same way, the proportions of insemination were comparedbetween males using logistic regression and the scores were basedon a linear regression using ANOVA.

3. Results

3.1. Larval development

There were no significant differences in pupation rates betweenthe different diets provided at a concentration of 1%. However,when Diet 3 was used at a concentration of 2%, the pupationrate was highest with 28% of larvae successfully transforming intopupae (Table 1). Overall, the emergence rates were marginallyhigher when diets were used at a concentration of 2%, with Diets2 and 3 providing the highest emergence rate of all treatmentswith 23–25% of larvae successfully completing their developmentinto adults (Table 1). When larvae were provided with higher con-centration, pupation occurred in a shorter time interval and wecollected almost all of the pupae in first 24 h after the first pupa-tion was observed. In this study, 73% and 78% of emergence wererecorded within 24 h of pupation when rearing at a treatment 2.2and treatment 3.2 respectively.

Kaplan–Meier curves drawn for the time taken to adult emer-

gence (Fig. 1) showed that this period was higher in larvae providedwith concentration of 1% compared to 2% regardless of diet com-position (Table 1). Larvae reared with Diet 2 took a shorter timeto emerge than those reared on either Diets 1 and 3, regardless

gambiae fed with six treatments.

Duration in day Wing lengths

8.96 ± 0.12a 2.499 ± 0.013ac 7.30 ± 0.09b 2.619 ± 0.031cb 7.44 ± 0.14b 2.462 ± 0.035abd 5.99 ± 0.06c 2.556 ± 0.018c

8.70 ± 0.15a 2.42 ± 0.025b 6.81 ± 0.06d 2.584 ± 0.02c

cantly different (p < 0.05; quasibinomial separation test).

Tropica 132S (2014) S96–S101 S99

opmt

fmw[Cl(l

aos

3

lvdcdotp3

((rtwvpp

p(lnrh

oTcm(dNh(3

3

fand(

Fig. 2. Distribution of the mean number of females inseminated per day by eachgroup of male related to the food treatment: five replicates of this test were per-formed by diet.

Table 2Comparison of the average scores of insemination between male.

Treatments Mean score/24 h Lifespan (day)

1.2 0.93 ± 0.03a �192.2 1.83 ± 0.01b 143.2 1.06 ± 0.04a �19

Means (based on five replicates) within a row followed by the same letter are notsignificantly different (p = 0.05; ANOVA test).

G.A. Yahouédo et al. / Acta

f concentration (p < 0.001, RR = 2.24, CI95% [1.68; 2.98] for 1%, and < 0.001, RR = 6.35, CI95% [4.88; 8.26] for 2%). The fastest develop-ent was achieved by using Diet 2 at 2%, with mean development

ime emergence of 5.99 days (Table 1).The relative chance (probability) of obtaining adult emergence

or each treatment (type of diet and each concentration) was deter-ined. Larval were 2.18 time more likely to emerge as adultsith treatment 2.1 than with treatment 1.1 (p = 0.0001, CI95%

1.64–2.91]) or treatment 3.1 (2.57 times more likely, p = 0.0001,I95% [0.28–0.52]). In the same line, there were 6.57 times more

ikely to emerge with treatment 2.2 than with treatment 1.2p = 0.0001, CI95% [5.08–8.50]) and treatment 3.2 (3.5 times moreikely p = 0.0001, CI95% [2.75–4.44]).

These data clearly showed that the chances of adult emergencet each time point are higher in treatment 2 than 1 or 3, regardlessf concentration. Thus, larvae reared with treatment 2.2 had thehortest development time (5.99 days, Fig. 1).

.2. Male size and energy reserves

At a concentration of 1%, we observed that the average wingength of males provided with Diet 1 is higher than males pro-ided with Diet 3 (p = 0.03, CI95% [0.861–0.991]). But, no significantifference were observed when compared to that of Diet 2. At aoncentration of 2%, no difference was observed between the threeiets (Table 1). However, it appeared that the average wing lengthf males reared with a 2% concentration were significantly higherhan those reared with a 1% concentration for all diets (Diet 1:

= 0.001, CI95% [1.05; 1.21], Diet 2: p = 0.010, CI95% [1.02, 1.17], Diet: p = 0.0001, CI95% [1.09–1.26]).

No significant differences in either lipid or sugar reservesp = 0.1, ANOVA test) were detected between the treatmentsTable 3). Glycogen reserves were significantly higher in maleseared on treatment 3.1 compared to those in males reared withreatment 1.1 (p = 0.048) or treatment 2.1 (p = 0.001). Males rearedith treatment 3.2 accumulated more glycogen than those pro-

ided with treatment 2.2 (p = 0.013). We conclude that Diet 3rovides the highest content of this metabolite (glycogen) com-ared to other diets (Table 3).

Males reared with treatment 3.1 accumulated significantly morerotein than those reared with treatment 1.1 (p = 0.018) or 2.1p = 0.0001). At a concentration of 2%, larvae accumulated simi-ar protein levels (Table 3). According to all these results, there iso feeding regime that provided the best results in term of adulteserves (metabolites), however Diet 3 may provide to male theighest levels of metabolite reserves.

The total energy reserves of an adult male are defined as the sumf these metabolic reserves (proteins, sugars, glycogen and lipids).hus, the total energy of the males resulting from each diet wasompared between the various groups. Males reared with treat-ent 3.1 had more energy than those reared with treatment 1.1

p = 0.035) or 2.1 (p = 0.0001). However, there was no significantifference in energy reserves at a concentration of 2% (Table 3).evertheless, we observed that males reared with treatment 3.1ad more energy reserves than those reared with treatment 2.2p = 0.013). There was no significant difference between treatment.1 and 3.2 (p = 0.094).

.3. Insemination test

Overall, a total of 2250 females were dissected to determine theemales which were scored inseminated (as positive) or not (neg-

tive: no sperm present in spermathecae). The variable “diet” didot explain the variability in the number of females inseminated byifferent males (p = 0.482, df = 2). However, the variables “period”p = 0.0001) and “lifespan” of the adult male (p = 0.060) were used

Fig. 3. Mean of the total number of females inseminated by all males of each dietgroup with Standard Error (SEM).

in our model to explain the phenomenon. Thus, more the num-ber of the females inseminated by a male, the lower is its lifespan(p = 0.0001, OR = 0.794, CI95% [0.740–0.847]). We also presented themean of females inseminated by each group of males/day duringthe fifteen days (Fig. 2). Furthermore, the mean scores of insemi-nation of male mosquitoes reared with treatment 1.2 or 3.2 weresignificantly lower than those reared with treatment 2.2 (p = 0.005)(Table 2). The sum of females inseminated in 15 days of lifespans bymales of treatment 2.2 was almost equal those reared with treat-ment 3.2 and higher than that of to 1.2 (Fig. 3) while these males(1.2) lived more than 19 days. We did not have enough males fromthe concentration of 1% having the same age for all diets in orderto synchronize the test of insemination.

4. Discussion

In this study, we evaluated three larval diets and the optimalconcentration for rearing Anopheles gambiae mosquitoes. Thesediets were specifically designed to be used for mass rearing, withthe aim of providing adequate nutritional components to optimize

S100 G.A. Yahouédo et al. / Acta Tropica 132S (2014) S96–S101

Table 3Value of several metabolites assessed [mean (J/mm3) ± SE] of adult males for each diet and it doses.

Treatment Metabolites

Lipids Sugar Protein Glycogen Energy

Control 2.44 ± 0.22a 67.89 ± 2.73a 86.97 ± 2.68a 64.96 ± 1.84a 222.30 ± 7.13a1.1 1.38 ± 0.15b 61.57 ± 1.69b 79.41 ± 3.72b 60.17 ± 1.32b 202.53 ± 6.02b1.2 2.10 ± 0.13c 64.77 ± 2.12ab 77.42 ± 2.61b 62.40 ± 2.99ab 206.68 ± 7.44ab2.1 1.53 ± 0.24b 57.59 ± 3.08c 68.71 ± 3.46c 55.02 ± 2.74c 182.85 ± 8.93c2.2 1.72 ± 0.12c 63.55 ± 1.87ab 74.16 ± 3.28bc 58.72 ± 2.74bc 198.16 ± 6.17ab3.1 0.93 ± 0.12d 67.02 ± 1.72a 89.89 ± 1.50a 68.51 ± 2.62a 226.35 ± 5.38a3.2 2.01 ± 0.23c 63.11 ± 3.77ab 73.21 ± 3.57bc 69.25 ± 4.93a 207.59 ± 11.79ab

M gnific

rpBapo(

bemTAmooI23mw

wCBodb(nbirtlDwlTt2oltaiibt

f

eans (based on ten replicates) within a row followed by the same letter are not si

earing and adult insect quality from ingredients which are inex-ensive, globally available and of consistent quality (Parker, 2005;enedict et al., 2009). We expected high larval survivorship, rapidnd homogenous larval development, size homogeneity within theopulation, synchronicity of the onset of pupation and productionf high quality adults in terms of longevity, and mating capacityBellini et al., 2007; Medici et al., 2011).

Overall the data of the present study showed that feeding dou-le the amount of diet improved all rearing parameters appraisedxcept the total energy reserves of adult males reared with treat-ent 3.1, though this finding was not significant for all parameters.

hese results could be explained by a simple quantity effect.nother interesting observation was noted during the experi-ents. When larvae were fed at the higher concentration, pupation

ccurred in a shorter time interval and we collected almost allf the pupae in first 24 h after the first pupation was observed.n this study, 73% and 78% of emergence were recorded within4 h from pupae when rearing with treatment 2.2 and treatment.2, respectively. This would give a great practical advantage whenass-rearing mosquitoes for irradiation and release is needed orhen a homogeneous population is desirable.

The shortest development time (5.99 days) of adult mosquitoesas recorded when larvae were provided with treatment 2.2.onsidering the composition of Diet 1 and Diet 2, it is likely thatrewer’s Yeast and/or squid liver powder was/were the ingredientf Diet 2 facilitating a more rapid development. An analysis of theiet ingredients showed that squid liver powder, tuna meal andovine liver powder are rich in proteins, vitamins and fatty acidsDamiens et al., 2012), while in “Brewer’s Yeast” the major compo-ents (50–65%) are carbohydrates. Diet 2 would be recommendedecause a shorter development time would produce a shorter rear-

ng schedule for obtaining the males to be released and thus wouldeduce operational cost of mass rearing. In addition, the survivalo adulthood of larval provided with Diet 2 was higher than whenarval were fed with Diets 1 and 3. In contrast, no males reared withiet 2 lived more than 14 days compared to treatments obtainedith Diet 1 or Diet 3 where at least two males from both treatments

ived more than 19 days even though they did not inseminate.his result could be explained by the shorter larval developmentime, meaning that males from this treatment (obtained from Diet) did not accumulate as many reserves during larval stages. Thisbservation could explain by a trade-off between reproduction andifespan (Miyatake, 1997). A choice must thus be made accordingo the needs of a release program because there is no utility with

male released with a long lifespan but which is incompetent atnseminating females. Further study, integrating the regularity ofnsemination according to different larval feeding regimes. It will

e interested to explore the presence or not of the mating plug inhe assessment of the male capacity to inseminate.

The metabolic assay conducted on young male mosquitoesrom different diets and concentration treatments allowed us to

antly different (p = 0.05; ANOVA test).

highlight the low proportion of lipids in all groups of males, com-pared to the controls reared on standard commercial fish food.These observations suggested several hypotheses: (i) the malesprovided only with water for 24 h after emergence exhausted theirfat reserves under stress conditions. In 1990, a study conductedby Briegel (1990b) showed that over 70% of the larval fat reserveswere mobilized during food stress conditions in Anophelinae; or(ii) this metabolite would be more solicited during the emergenceprocess. A decrease in lipid reserves has already been noted in Cer-atitis capitata (Diptera: Tephritidae) after emergence and it takesup to six days before there is a reconstitution of the reserve inadults (Warburg and Yuval, 1996). These questions require com-plementary research to explain the low proportion of lipids in allnewly emerged males. The high level of reserves observed in malesfrom the treatment 3.1 could be explained by their longer develop-ment time relative to males from other treatments. In fact, the moreenergy for reproduction is stored as lipid and carbohydrate reservesduring the larval stage, the greater the development duration of anorganism is extended (Nisbet et al., 2000). The amount of energyinvested in growth and the rate at which growth occurs is one of thecompromises organisms make during development. Thus, for theSterile Insect Technique, it is important to take into account theseparameters in young males for release activities because it is notideal to release males that after a few days will have much lowerreserves than their wild counterparts in the field.

5. Conclusion

This study allowed us to assess the performance of three larvaldiets provided by IAEA, used at different concentrations, throughthe measurement of several development parameters. We sug-gested through this study that treatment 2.2 with a density of1 L1/ml could be the best feeding regime for mosquito mass-rearing. This treatment produces male mosquitoes in short timeand they recorded the best insemination scores. However, the crite-ria of a better diet are not limited only to the larval developmenttime and insemination scores showed by the males. It is also rele-vant to take into account the parameter of lifespan and flight abilitythat were not investigated in details in the present study. Furtherwork needs to be done to find out the best combination of food andother rearing conditions that will provide the best fitness to themales to be released in the field.

Acknowledgements

This work was supported by a grant by IAEA research contracts

no. 16412/R0 to LSD and no. 14777/R0 to FS under the CRP G43002.Authors are grateful to L. Djossou for technical assistance duringlaboratory assays and mosquito collections and to C. Brengues formetabolic dosage assistance.

Tropi

R

A

A

A

B

B

B

B

B

B

B

B

C

C

C

C

C

C

D

D

D

G.A. Yahouédo et al. / Acta

eferences

lphey, L., Benedict, M., Bellini, R., Clark, G., Dame, D., Service, M., Dobson,S., 2010. Sterile-insect methods for control of mosquito-borne diseases:an analysis. Vector Borne Zoonotic Dis. (Larchmont, N.Y.) 10, 295–311,http://dx.doi.org/10.1089/vbz.2009.0014.

rmbruster, P., Hutchinson, R., 2002. Pupal mass and wing length as indicators offecundity in Aedes albopictus and Aedes geniculatus (Diptera: Culicidae). J. Med.Entomol. 39, 699–1403.

meneshewa, B., Service, M.W., 1996. The relationship between female body sizeand survival rate of the malaria vector Anopheles arabiensis in Ethiopia. MedVet Entomol 10, 170–172.

aumhover, A.H., 2002. A personal account of developing the sterile insect techniqueto eradicate the screwworm from Curacao, Florida and the Southeastern UnitedStated. Florida Entomol. 85, 666–673 http://www.fcla.edu/FlaEnt/fe85p666.pdf

ellini, R., Calvitti, M., Medici, A., Carrieri, M., Celli, G., Maini, S., 2007. Use of thesterile insect technique against Aedes albopictus in Italy: first results of a pilottrial. In: Vreysen, M.J.B., Robinson, A.S., Hendrichs, J. (Eds.), Area-wide Control ofInsect Pests: From Research to Field Implementation. Springer, Dordrecht, TheNetherlands, pp. 505–515.

enedict, M.Q., Knols, B.G.J., Bossin, H.C., Howell, P.I., Mialhe, E., Caceres, C., Robinson,A.S., 2009. Colonisation and mass rearing: learning from others. Malar. J. 8 (Suppl.2), S4.

enedict, M.Q., Robinson, A.S., 2003. The first releases of transgenic mosquitoes: anargument for the sterile insect technique. Trends Parasitol. 19 (8), 349–355.

oggs, C., Freeman, K., 2005. Larval food limitation in butterflies:effects on adult resource allocation and fitness. Oecol 144, 353–414,http://dx.doi.org/10.1007/s00442-005-0076-6.

riegel, H., 1990a. Fecundity, metabolism, and body size in Anopheles (Diptera: Culi-cidae), vectors of malaria. J. Med. Entomol. 27, 839–889.

riegel, H., 1990b. Fecundity, metabolism, and body size in anopheles (Diptera:Culicidae), vectors of malaria. J. Med. Entomol. 27, 839–850.

riegel, H., 1990c. Metabolic relationship between female body size, reserves, andfecundity of Aedes aegypti. J. Insect Physiol. 36, 165–337.

aceres, C., Cayol, J.P., Enkerlin, W.R., Franz, G., Hendrichs, J., Robinson, A.S., 2004.Comparison of Mediterranean fruit fly (Ceratitis capitata) (Tephritidae) bisexualand genetic sexing strains: development evaluation and economics. In: Barnes,B.N. (Ed.), Proceedings, Symposium: 6th International Symposium on Fruit Fliesof Economic Importance. 6–10 May 2002, Stellenbosch, South Africa. Isteg Sci-entific Publications, Irene, South Africa, pp. 367–381.

atteruccia, F., Crisanti, A., Wimmer, E., 2009. Transgenic technologies to inducesterility. Malar. J. 8 (Suppl. 2), http://dx.doi.org/10.1186/1475-2875-8-s2-s7.

harlwood, J., 1996. Biological variation in Anopheles darlingi Root. Memórias doInstituto Oswaldo Cruz 91, 391–399.

harlwood, J., Pinto, J., Sousa, C.A., Madsen, H., Ferreira, C., do Rosario, V.E., 2003. Theswarming and mating behaviour of Anopheles gambiae s.s. (Diptera: Culicidae)from São Tomé Island. J. Vector Ecol. 27, 178–183.

harlwood, J.D., Jones, M.D.R., 1979. Mating behaviour in the mosquito, Anophelesgambiae s.l. I. Close range and contact behaviour. Physiol. Entomol. 4, 111–120.

ohuet, A., Dia, I., Simard, F., Raymond, M., Fontenille, D., 2004. Popula-tion structure of the malaria vector Anopheles funestus in Senegal basedon microsatellite and cytogenetic data. Insect Mol Biol. 13, 251–259,http://dx.doi.org/10.1111/j.0962 1075.2004.00482.x.

abiré, K., Diabaté, A., Djogbenou, L., Ouari, A., N’Guessan, R., Ouédraogo,J.-B., Hougard, J.-M., Chandre, F., Baldet, T., 2008. Dynamics of mul-tiple insecticide resistance in the malaria vector Anopheles gambiae ina rice growing area in South-Western Burkina Faso. Malar. J. 7, 188,http://dx.doi.org/10.1186/1475-2875-7-188.

amiens, D., Benedict, M., Wille, M., Gilles, J., 2012. An inexpensive and effectivelarval diet for Anopheles arabiensis (Diptera: Culicidae): eat like a horse, a bird,

or a fish? J. Med. Entomol. 49, 1001–1011.

iabate, A., Baldet, T., Brengues, C., Kengne, P., Dabire, K., Simard, F., Chandre, F.,Hougard, J., Hemingway, J., Ouedraogo, J., Fontenille, D., 2003. Natural swarmingbehaviour of the molecular M form of Anopheles gambiae. Trans. R. Soc. Trop.Med. Hyg. 97, 713–719.

ca 132S (2014) S96–S101 S101

Diabaté, A., Dabire, R., Kengne, P., Brengues, C., Baldet, T., Ouari, A., Simard, F.,Lehmann, T., 2006. Mixed swarms of the molecular M and S forms of Anophe-les gambiae (Diptera: Culicidae) in sympatric area from Burkina Faso. J. Med.Entomol. 43, 480–483.

Djogbénou, L., Pasteur, N., Akogbeto, M., Weill, M., Chandre, F., 2010. Insecticideresistance in the Anopheles gambiae complex in Bena nationwide survey. Med.Vet. Entomol. 253, 256–267.

Fernandes, Briegel, L.H., 2005. Reproductive physiology of Anopheles gambiae andAnopheles atroparvus. J. Vector Ecol. 30, 11–37.

Foster, W., Takken, W., 2004. Nectar-related vs. human-related volatiles:behavioural response and choice by female and male Anopheles gambiae(Diptera: Culicidae) between emergence and first feeding. Bull. Entomol. Res.94, 145–202.

Hawley, W., 1985. A high-fecundity aedine: factors affecting egg production of thewestern treehole mosquito, Aedes sierrensis (Diptera: Culicidae). J. Med. Entomol.22, 220–225.

Helinski, M.E.H., El-Sayed, B., Knols, B.G.J., 2006. The Sterile Insect Technique: canestablished technology beat malaria? Entomol. Ber. 66, 13–33.

Hogg, C.J., Thomson, M.C., Hurd, H., 1996. Comparative fecundity and associatedfactors for two sibling species of the Anopheles gambiae complex occurring sym-patrically in The Gambia. Med. Vet. Entomol. 10, 385–391.

Klassen, W., 2009. Introduction: development of the sterile insecttechnique for African malaria vectors. Malar. J. (Suppl. 2), I18,http://dx.doi.org/10.1186/1475-2875-8-S2-I1.

Kooijman, S., 2000. Dynamic Energy and Mass Budgets in Biological Systems.Cambridge University Press.

Lyimo, E., Takken, W., 1993. Effects of adult body size on fecundity and the pre-gravidrate of Anopheles gambiae females in Tanzania. Med. Vet. Entomol. 7, 328–360.

Medici, A., Carrieri, M., Scholte, E.J., Maccagnani, B., Dindo, M.L., Bellini, R., 2011.Studies on Aedes albopictus larval mass-rearing optimization. J. Econ. Entomol.104, 266–273.

Miyatake, T., 1997. Genetic trade-off between early fecundity and longevityin Bactrocera cucurbitae (Diptera: Tephritidae). Heredity 78 (Pt 1),93–100.

Ng’habi, K., John, B., Nkwengulila, G., Knols, B., Killeen, G., Ferguson, H., 2005. Effectof larval crowding on mating competitiveness of Anopheles gambiae mosquitoes.Malar. J. 4, 49, http://dx.doi.org/10.1186/1475-2875-4-49.

Nisbet, R., Muller, E., Lika, K., Kooijman, S., 2000. From molecules to ecosys-tems through dynamic energy budget models. J. Anim. Ecol. 69, 913–1839,http://dx.doi.org/10.1111/j.1365-2656.2000.00448.x.

WHO, 2001. L’OMS publie des lignes directrices en matière de culture de la planteessentielle utilisée dans les médicaments antipaludiques. Centre des médias.

Parker, A.G., 2005. Mass-rearing for sterile insect release. In: Dyck, V.A., Hendrichs,J., Robinson, A.S. (Eds.), Sterile Insect Technique. Principles and Practice in AreaWide Integrated Pest Management. Springer, Dordrecht, The Netherlands, pp.209–232.

Puggioli, A., Balestrino, F., Damiens, D., Lees, R.S., Soliban, S.M., Madakacherry, O.,Dindo, M.L., Bellini, R., Gilles, J.R.L., 2013. Efficiency of Three Diets for LarvalDevelopment in Mass Rearing Aedes albopictus (Diptera: Culicidae). J. Med.Entomol 50 (4), 819–825.

Rathor, H., Toqir, G., Reisen, W., 1980. Status of insecticide resistance in anophelinemosquitoes of Punjab Province, Pakistan. Southeast Asian J. Trop. Med. PublicHealth 11, 332–372.

Reim, C., Teuschl, Y., 2006. Size-dependent effects of larval and adult food avail-ability on reproductive energy allocation in the Yellow Dung Fly. Funct. Ecol.,http://dx.doi.org/10.1111/j.1365-2435.2006.01173.x.

Rivero, A., Agnew, P., Bedhomme, S., Sidobre, C., Michalakis, Y., 2007. Resource deple-tion in Aedes aegypti mosquitoes infected by the microsporidia Vavraia culicis.Parasitology 134, 1355–1417, http://dx.doi.org/10.1017/s0031182007002703.

Warburg, M.S., Yuval, B., 1996. Effects of diet and activity on lipid lev-

els of adult Mediterranean fruit flies. Physiol. Entomol. 21, 151–309,http://dx.doi.org/10.1111/j.1365-3032.1996.tb00848.x.

Weidhaas, D., 1962. Chemical sterilization of mosquitoes. Nature 195, 786–787.Yuval, B., Bouskila, A., 1993. Temporal dynamics of mating and predation in mosquito

swarms. Oecol 95, 65–134.