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International Journal of Tropical
Insect Science
e-ISSN 1742-7592
Int J Trop Insect Sci
DOI 10.1007/s42690-020-00134-7
Optimization of Aedes albopictus rearing
procedures for combined sterile insect
techniques (SIT) and Wolbachia-based
laboratory studies in Sri Lanka
N.D.A.D.Wijegunawardana,
Y.I.N.Silva Gunawardene,
W.Abeyewickreme,
T.G.A.N.Chandrasena,
R.S.Dassanayake, et al.
1 23
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ORIGINAL RESEARCH ARTICLE
Optimization of Aedes albopictus rearing procedures for combined
sterile insect techniques (SIT) and Wolbachia-based laboratory
studies in Sri Lanka
N. D. A. D. Wijegunawardana
1,2
&Y. I. N. Silva Gunawardene
1
&W. Abeyewickreme
3
&T. G. A. N. Chandrasena
4
&
R. S. Dassanayake
5
&A. Manamperi
1
Received: 28 November 2019 / Accepted: 5 March 2020
#African Association of Insect Scientists 2020
Abstract
The establishment of a laboratory colony is an essential first step for conduction of laboratory studies on the biology and control
of Aedes albopictus mosquitoes. Therefore, with the objective of generating high quality mosquitoes as research material for
utilization in ongoing vector control studies and to disseminate authenticated, high-quality Ae. albopictus mosquito rearing
information to the research community, maintenance of an Ae. albopictus mosquito colony was initiated at the Molecular
Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka. A self-mating colony was established from eggs of
the F1 progeny of individuals collected as free-living larvaein Narahenpita (Western Province), Sri Lanka. The mean temperature
of 28 °C (± 2 °C) and relative humidity of 80% (± 5%) was constantly maintained inside the insectary. Lighting was provided by
fluorescent lights, regulated with 12:12 h continuous dark and light period. Pest insects were controlled manually. Mosquitoes
were maintained on bovine blood provided via an artificial membrane feeding system and a continuous supply of 10% sugar
supplements. Larvae were maintained in deoxygenate water and fed with International Atomic Energy Agency (IAEA) recom-
mended diet of tuna meal, bovine liver powder, and brewery yeast in a ratio of 37.5:27:10.5 in 1 L. Data on fecundity, fertility,
larvae death, pupation, adult emergence, adult mosquito longevity were recorded. Adhering to bio-safety, all discarded materials
were boiled thoroughly and incinerated if required. This report on the establishment and maintenance of a laboratory colony of
Ae. albopictus will be of value for identifying the critical requirements essential under artificial conditions.
Keywords Aedes albopictus .Laboratory colonization .Optimum rearing conditions
Introduction
The mosquito genus Aedes consist of over 950 species which
are native to temperate and tropical habitats worldwide (Yi
et al. 2014). Forty eight different species of Aedes were found
to prevail in Sri Lanka in 2013 (Sirisena and Noordeen 2013).
Of them, Ae. albopictus, a secondary vector of dengue in Asia
(Christofferson 2015) is the predominant mosquito species in
Sri Lanka.
Ae. albopictus has a wide geographical distribution, as it is
particularly resilient, and can survive in both rural and urban
environments (Benedict et al. 2007). The mosquito’seggsare
highly resistant and can remain viable throughout the dry sea-
son (Cunze et al. 2016). This mosquito species can also sur-
vive in cooler temperate regions of Europe (Cunze et al.
2016).ThereforeinadditiontomostAsiancities,Ae.
albopictus is prevalent in large parts of the United States,
Brazil and Europe (Lambrechts et al. 2011; Delatte et al.
2009; Medlock et al. 2012;Carvalhoetal.2014).
Approaches to reduce dengue infection include reduction
of mosquito abundance, prevention or minimizing mosquito-
human contact, genetic manipulation of vector mosquitoes to
reduce vector efficacy and vaccines (Eisen et al. 2009; Gubler
*Y. I. N. Silva Gunawardene
nilminisg@kln.ac.lk
1
Molecular Medicine Unit, Faculty of Medicine, University of
Kelaniya, Ragama, Sri Lanka
2
Department of Bioprocess Technology, Faculty of Technology,
Rajarata University of Sri Lanka, Anuradhapura, Sri Lanka
3
Department of Parasitology, Faculty of Medicine, General Sir John
Kotelawala Defence University, Colombo, Sri Lanka
4
Department of Parasitology, Faculty of Medicine, University of
Kelaniya, Ragama, Sri Lanka
5
Department of Chemistry, Faculty of Science, University of
Colombo, Colombo, Sri Lanka
International Journal of Tropical Insect Science
https://doi.org/10.1007/s42690-020-00134-7
Author's personal copy
1988). In the absence of a satisfactory vaccine for dengue, the
only available control strategies are mosquito based. (http://
www.who.int/immunization/research/development/dengue_
q_and_a/en/,CDC2017). Thus, investigations for more
effective mosquito control methods has become a priority.
Scientific experiments related to the mosquito biology and
control require laboratory reared uninfected mosquitoes for
testing. Therefore, rearing facilities for mosquitoes need to
be established at the onset of such investigations with main-
tenance of quality to guarantee a high production rate of
healthy mosquitoes to be utilized in research. The combined
Sterile Insect Technique (SIT) and Wolbac h ia-based approach
(Incompatible Insect Technique IIT) are new tools that have
much potential in the integrated vector management strategies
planned against Ae. albopictus mosquitoes in Sri Lanka
(Wijegunawardana et al. 2017). These methods require a con-
tinuous supply of massive quantities of laboratory bred mos-
quitoes for interventions with Wol b ach ia and or ionizing radi-
ation. Thus large scale mosquito rearing facilities need to be
established for application of the above vector control
methods. In addition, the success of these methods are largely
dependent on the survival, dispersal and mating competitive-
ness of the treated male mosquitoes with those in the target
zone. If these fitness parameters are comparable or better than
those of wild mosquitoes, the suppression of the wild mosqui-
to population could be achieved by way of reproductive in-
compatibility in the long term (Benelli et al. 2016). Therefore,
optimization of Ae. albopictus rearing facility is a primary
requirement for healthier mosquitos. Secondly, optimization
of rearing facilities and procedures facilitates synchronized
development of mosquitos which has a greater production
value in terms of, cost and time. Thus this study was aimed
at gaining information on the best practices and procedures
required for optimization of Ae. albopictus rearing and we
report the optimized protocol with the intention of sharing
the knowledge within the research community working in a
similar field and for those intending to set up insectary facility
in their institutions. The optimized protocol highlighted in this
article provide authenticated, high-quality Ae. albopictus mos-
quito rearing information and technology.
Materials and methods
Insectary facilities and environmental conditions
All research work outlined in this manuscript was undertaken
at the insectary of the Molecular Medicine Unit (MMU),
Faculty of Medicine, University of Kelaniya, Sri Lanka.
From the onset, the internal environment of the insectary
was carefully regulated to better suit the colonization of Ae.
albopictus mosquito. The mean temperature of 28 °C (± 2 °C)
and relative humidity (RH) of 80% (± 5%) was constantly
maintained inside the insectary with air conditioning and a
humidistat for auto-control of the humidifier (Deffensor
D505, Cat. No., OT2099B, Condair, USA). Lighting was pro-
vided by fluorescent lights, controlled with a 12:12 h contin-
uous scoto and photo periods (Higgs and Beaty 1996). Pest
insect were eliminated manually ensuring the essential ab-
sence of ants and cockroaches. Application of insecticides or
repellents were not allowed within the insectary for any pur-
pose. This was done to avoid any harm to the mosquito colo-
nies either directly or by contamination with toxicants
transported by pests. An optimally functional adult mosquito
trap was placed inside the insectary and was monitored for
released mosquitoes. The level of cleanliness inside the insec-
tary was consistently maintained. Written guidelines were giv-
en to each person assigned to a particular task inside the in-
sectary facility.
Insectary operations
Direct insectary operations included rearing of Ae. albopictus
larvae, pupae and adult for routine colony maintenance; facil-
itate egg laying for adults after blood feeding; egg collection,
counting and hatching. Number of Ae. albopictus eggs on
each egg paper (Seed germination papers/Filter paper, Grade
6 S/N, Cat. FT-2-314-580,580) was counted and recorded be-
fore starting the egg hatching process. Preparation of hatching
bottles were done on the afternoon of the day prior to the start
of egg hatching. Hatching bottles were filled with boiled dis-
tilled water and the lids were immediately tightened for com-
plete elimination of oxygen from the water. After allowing the
hatching bottles to cool to room temperature, egg papers were
sub-merged in the hatching bottle for a maximum of 2 days
with regular inspection for egg hatching.
After 2 days of hatching, the larvae that emerged from the
eggs were put into larvae rearing trays (size W400 x D300 x
H80 mm, plastic, white, heat resistant up to 70 °C) half filled
with de-chlorinated water. Larvae were fed with International
Atomic Energy Agency (IAEA) recommended diet of tuna
meal, bovine liver powder, and brewery yeast and vitamin in
a ratio of 37.5:27:10.5: g in 1 L up to 1 week (IAEA 2017).
Two types of larval diet supplementation methods were prac-
ticed under similar larval rearing conditions. The first method
consisted of supplements of IAEA standard larvae diet mix
1.5 μl per larvae up to pupation and the second method in-
cluded supplement of larvae diet according to following reg-
imen; 1.5 μl day one, 1.5 μl; day two, 1.55 μl; day three,
1.6 μl; day four, 1.65 μl and day five, 1.7 μl to facilitate
maximum larval growth and thereafter again decrease to stan-
dard volume of diet (1.5 μl/larvae) (Balestrino et al. 2014).
The trays were covered with fine nets in order to avoid egg-
laying by adult mosquitoes from different sources. Cultures of
mosquito larvae were monitored daily and precautions were
taken to avoid the growth of a bacterioneuston layer, a thin
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organic surface microlayer that covers the water surface which
has a negative impact on oxygen intake leading to high mor-
tality rate of the larvae (Norkrans 1980). Therefore the
microlayers formed were regularly removed by scraping a
tissue paper across the water surface.
Pupation started after the fifth day of egg hatching and
ended by the 7th day. Number of pupa were counted and their
gender was recorded. Sex determination of pupa was done
using size variation. For purposes of quality control micro-
scopic identification was performed among randomly selected
samples and length and width of pupal cephalothorax were
measured and recorded (OPTIKA Srl, V2.0, Italy). At the
same time any identifiable phenotypic variations on pupae
were recorded. An equal number of male and female pupa
(minimum of 150 and maximum of 500 from each sex) were
counted and put into pupa cup and kept inside the adult Bug
Dorm cages (30 × 30 × 30 cm
3
,E6099,Bioquip)upto2days
allowing the adults to emerge.
After emergence adults were fed on a 10% sucrose solu-
tion. As females required a blood meal for maturation and egg
laying, blood feeding was started from 4th day onwards with
bovine blood. Feeding was done for about 1 h per cage with
1000 mosquitoes with a 1:1 male female ratio using an artifi-
cial membrane feeder. Egg laying cups were kept 2 days after
the blood feeding and collection of egg laying papers were
done on the fourth day post-feeding. Egg papers were dried
for a minimum of 3 days at room temperature prior to the start
of the hatching process. Adult mosquito cages were blood fed
every 4th day after emergence from pupa and for purposes of
quality control each adult cage was blood fed only thrice and
there after supplementation with 10% sugar solution was con-
tinued until the death of all adult mosquitoes.
Records on larval feeding, larvae tray maintenance and
cleaning charts, adult feeding (both sugar solution and blood)
and insectary cleaning were maintained on a daily basis. Data
recorded included rates of egg laying, egg hatching, larvae
death, pupation, adult emergence, egg laying and adult mos-
quito death with respect to sex and time difference.
Appropriate safety precautions were followed with regard to
insectary waste disposal. For purposes of bio-safety all
discarded material from larvae trays, egg laying cupsand adult
cages were thoroughly boiled to facilitate total destruction of
contaminant mosquito eggs in the discarded material. All oth-
er infectious materials such as cotton pads soaked with bovine
blood, feeding membranes and gloves were incinerated. For
safety and security reasons insectary access was restricted to
authorized staff who were trained in mosquito handling and
waste disposal, etc.
Data analysis
Frequency data were angle transformed (arcsine sqrt) while
count data log transformed (Log10 (n + 1) before testing for
normality (Anderson-Darling test) and homogeneity of vari-
ances (Levene’s test) prior to statistical analysis. Differences
in measured parameters among Ae. albopictus (e.g, pupae
size) were analyzed using one-way ANOVAs. The General
Linear Models were used to evaluate the impact of larval diet
on growth parameters (e.g., pupae size). Means were separat-
ed by Tukey’s Post hoc test. For pairwise comparison, the
Student ttests or the nonparametric Mann-Whitney U tests
were used. All statistical analyses were performed using
Statistical Package for the Social Sciences (SPSS) software
(IBM® SPSS® Statistics) with alpha level of 0.05. Unless
otherwise stated, back-transformed values (mean and 95%
confidence interval (CI) are presented in the text and figures
to aid interpretability.
Results
Performance indicators for Ae. Albopictus mosquito
colony from G#1 to G#14
Performance indicators (egg production and hatching rates
and pupation rates) of the up scaled colony of Ae. albopictus
were compared for each generation up to the 14th generation
with those prior to upscaling of colony maintenance as shown
in in Table 1. Accordingly, there was a significant effect on the
rates of the egg laying, egg hatching and pupation up to the G
# 14 for the laboratory bred Ae. albopictus colony after the
upscaling with standard rearing practices.
Female fecundity and egg viability (fertility)
Fecundity and fertility rates for each generation of the Ae.
albopictus isgiveninFig.1. Accordingly, fecundity varied
from 25 to 110 within a generation from the beginning of
the first generation, while giving an average of 42. After
the 6th generation onwards the mean difference of the fe-
cundity between the generations was reduced up to a range
of 48 to 59 (Fig. 1). Similarly, fertility rate increased ini-
tially from 77% to 98% within a maximum time period of
24 h. For better identificationoffirststage(L1)larvae
hatching bottles were kept for a further 24 h without any
disturbances. However, a significant difference in egg
hatching/fertility rates were not observed between each
generation (p=0.000)asdepictedinFig.1.
Rate of larvae survival and pupation
High survival rates in transition from larvae to pupa was
achieved with many challenges since this step depended on
both the environmental conditions and larval diet regi-
mens. Initially a small number of larvae per tray were used
and diet regimen and tray cleanliness were regularly
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maintained. Even though the number of dead larvae per
tray decreased from 40 to 7 it again increased when the
number of larvae reared per tray and number of days that
the larvae tray was maintained increased (Fig. 2). The red
colour circles in Fig. 2indicate the number of larvae reared
in each tray with the corresponding value along with the
number of day’s larvae tray was maintained until the pu-
pation of final active larvae. As a result of careful moni-
toring of these data finally 94% larval survival rate was
achieved by maintaining 1000 larvae per tray up to maxi-
mum of 7 days.
Pupation was initiated 5 days after larval emergence and it
took almost another 5 days to achieve 100% pupation.
Following optimization of larval diet and larval tray mainte-
nance time, 100% pupation was achieved successfully within
5 to 6 days after larval emergence. Rate of pupation ranged
from 0.49 to 1 from 1st generation to 14th generation, while
recording an average pupation rate of 0.84. Pupation rate was
more stable after the 4th generation and resulted in an average
pupation rate of 0.90 (Table 2) within the first 6 days after
larval emergence. Difference of both the rate and frequency of
male and female pupation was not statistically significant
1234567891011121314
Fecundity 42 39 54 34 44 35 48 56 51 52 58 55 52 59
Fertility 77 80 77 89 77 92 85 88 97 89 89 90 87 98
Rate of female fecundity &
egg viability (fertility)
Fig. 1 Rate of female fecundity
and egg viability (fertility) with
respect to the corresponding gen-
eration of the Ae. albopictus
mosquito colony
Table 1 Results of tests between-
subject’s effects of rates of egg
laying, hatching and pupation for
the Ae. albopictus mosquito
colony for each of 14 generations
Source Dependent Variable Type III Sum
of Squares
df Mean
Square
FSig.
Corrected Model EGGs 80,835.539
a
13 26,945.180 34.867 .000
Hatch rate 2.490
b
13 .830 9.147 .000
Rate of pupation .922
c
13 .307 5.724 .001
Intercept EGGs 895,731.234 1 895,731.234 1159.072 .000
Hatch rate 101.962 1 101.962 1123.820 .000
Rate of pupation 128.706 1 128.706 2397.573 .000
Treatment EGGs 80,835.539 13 26,945.180 34.867 .000
Hatch rate 2.490 13 .830 9.147 .000
Rate of pupation .922 13 .307 5.724 .001
Error EGGs 265,070.432 343 772.800
Hatch rate 31.120 343 .091
Rate of pupation 18.413 343 .054
Total EGGs 1,261,248.000 347
Hatch rate 161.754 347
Rate of pupation 207.330 347
Corrected Total EGGs 345,905.971 346
Hatch rate 33.609 346
Rate of pupation 19.335 346
a
R Squared = .234 (Adjusted R Squared = .227)
b
R Squared = .074 (Adjusted R Squared = .066)
c
R Squared = .048 (Adjusted R Squared = .039)
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within and between generations (Fig. 3). However, there was a
significant difference (H = 6.300, df = 1, P< 0.01) between
sexes for pupal cephalothorax measurements. Female speci-
mens were larger (mean 3.29 mm ± SD 0.12) than males
(2.58 mm ± 0.60). The impact of the larval diet regimens on
size variation between two sexes were not significantly differ-
ent (P> 0.05) with the two larvae diet supplement methods
and the correlation was not statistically significant at a pvalue
of 0.05.
The rate of adult emergence and survival
A 100% survival rate of pupated larvae up to adult emergence
was achieved within 2 days following the onset of pupation.
With the increase of larvae diet, rates of female emergence
increased more than the males from the 6th generation on-
wards as shown in Table 2. Adult survival rate up to the
12th day was 95.5%. There was no significant difference of
adult longevity between the sexes up to the first 12 days of
emergence. However, the approximate life span for males
(~17 days) was lower than the females (~ 25 days) and the
mortality was regular throughout all generations (G1 to G14).
Discussion
In agreement with others the hatching of Ae. albopictus mos-
quito eggs appears to be induced under anoxic conditions
generated by boiled water than normal tap water (mean hatch
rate of A. albopictus eggs in anoxic water was 87% (n=14)
and normal tap water was 63% (n = 14) (Borg and Horsfall
1953; Judson 1960; Fallis and Snow 1983). Thus, in large
scale laboratory rearing of Ae. albopictus an anoxic hatching
medium is recommended which would synchronize the egg
hatching within the first 24 h after its onset. An anoxic condi-
tion in the hatching medium can be easily achieved by boiling
the water as described in this report. Alternative methods such
as bubbling nitrogen gas through the water (Fallis and Snow
1983), adding ascorbic acid (Schwan and Anderson 1980;
Mulla and Chaudhury 1968), yeast (Morlan et al. 1963;
Farnesi et al. 2009) or Nutrient Broth (Bellini et al. 2007)to
the hatching medium has also been practiced.
Female fecundity (mean ± SE) was at an acceptable level
for sustain the mosquito colony (48 ± 3 eggs per female) with
a mean value ranging from 33 to 58. This was comparable to
other studies investigating the first gonotrophic cycle of the
mosquito (mean value ranging from 42 to 143 eggs per female
(Deng et al. 2012; Hawley 1988) but was higher than that
reported by Balestrino et al. 2014 (13 ± 1 eggs per female).
An interplay of several factors have been associated with the
high variations in female fecundity. These factors include the
size of mosquito (modulated by the colonization process or
the larval rearing conditions), blood meal source, blood feed-
ing method, availability of carbohydrates, age of mosquito,
mating conditions and suitability of oviposition sites (Deng
et al. 2012; De Jesus and Reiskind 2016). The low variation in
female fecundity observed in the current study as compared to
others confirmed that the rearing conditions between genera-
tions were more or less constant.
Table 2 Rate of pupation, adult emergence and survival with respect to
each generation of the Ae. albopictus laboratory mosquito colony
Generation Rate of (%)
Pupation Adult emerge Adult survival
Male Female Male Female
1 0.49 0.51 0.49 0.96 0.9
2 0.68 0.6 0.4 0.94 0.94
3 0.63 0.54 0.46 0.97 0.94
4 0.87 0.61 0.39 0.97 0.97
5 0.91 0.52 0.48 0.94 0.95
6 0.81 0.42 0.58 0.94 0.93
7 0.84 0.48 0.52 0.99 0.96
8 0.95 0.5 0.5 0.93 0.93
9 0.94 0.4 0.6 0.86 0.95
10 0.99 0.43 0.57 0.88 0.91
11 1 0.46 0.5 4 0.93 0.89
12 0.83 0.48 0.52 0.96 0.86
13 0.86 0.44 0.56 0.91 0.94
14 0.94 0.45 0.55 0.95 0.96
0
10
20
30
40
50
123456789101112131415161718
Average no. of dead
larvae per tray
No. of days
500
600
800
1000
Fig. 2 Average number of dead
larvae per tray with respect to the
number of days of Ae. albopictus
larval rearing under the insectary
rearing condition. Further red
colour circles highlighted the
fluctuation of dead larvae count
along with the variation of the
total number of larvae reared per
larvae tray
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The rate of pupation, adult emergence and adult sur-
vival was also at an acceptable rates for sustainable pro-
duction of mosquito colony with the optimized rearing
condition in the current study. However, there was a sex-
ual size dimorphism in Ae. albopictus mosquito pupae.
Literature indicates that this may be an influence of the
larvae diet (Castro et al. 1994; Consoli and Lourenço-de-
Oliveira 1994;Couretetal.2014). Since pupal stage is a
non-feeding stage, the diet during the larval period criti-
cally influences pupal size and development (Consoli and
Lourenço-de-Oliveira 1994;Couretetal.2014). As the
pupal size is regarded as an indicator of the mosquito
gender (IAEA 2017) and thus used as a parameter in the
application of sex separation required for SIT and
Wolb a chi a based approach an attempt was taken to mea-
sure the impact of larvae diet on pupal size. However,
with the two larvae diet regimens tested a significant cor-
relation was not observed. Therefore, continuation with
the standard IAEA diet mix with 1.5 ml/larvae is recom-
mended until a cheaper comparable IAEA diet option is
recommended.
Absence of a significant difference in adult longevity be-
tween adult males and females within first 12 days of emer-
gence indicate the existing insectary conditions are favorable
for rearing Ae. albopictus in Sri Lanka. Thus, this article will
be of assistance in identifying the critical requirements for the
establishment and maintenance of a colony of Ae. albopictus
under artificial conditions in Sri Lanka and in other similar
settings.
Acknowledgements Financial support received through NRC TO 14/04,
IAEA RAS 5047 and WHO/TDR grant HQTDR1409931 (TIMS ID:
B40098) are greatly acknowledged.
Compliance with ethical standards
Conflict of Interest The authors declare that they have no conflict of
interest.
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