1
Host plant relationships, temperature tolerance and mating compatibility studies
in Bactrocera cucurbitae and Ceratitis rosa
(Contract No. 16072/R0)
Part of IAEA/FAO Co-ordinated Project: Resolution of Cryptic Species
Complexes of Tephritid Pests to Overcome Constraints to SIT
Application and International Trade
2nd PROGRESS REPORT
icipe
P. O. Box 30772, 00100 GPO, Nairobi, Kenya
Chief Scientific Investigator: Dr. S. Ekesi
June 28, 2013
2
Program of work
The contract was awarded to cover the following program of work:
1. Establish Ceratitis rosa colonies and share materials with relevant CRP
participants.
2. Assess mating compatibility between lowland and highland populations of C.
rosa.
3. Assess the effect of temperature on development and survival of lowland and
highland populations of C. rosa.
4. Establish Bactrocera cucurbitae colonies from different hosts and habitats.
5. Catalogue B. cucurbitae host plants and carry out host preference studies,
preferably under field cage conditions.
During the period under review, the following progress was made with regard to the
stated objectives above.
Ceratitis rosa
1. Establish Ceratitis rosa colonies and share materials with relevant CRP
participants.
Background and rationale. In coastal Kenya, traditionally, Ceratitis rosa has been
reared from the following host plants: Psidium guajava (Myrtaceae), Monodora
grandidieri, Uvaria lucida (Annonaceae), Salacia elegans (Hippocrateaceae) and
Dryetes natalensis (Euphorbiaceae). All the listed host plants fruits between
September and December and effort to establish colonies of the coastal population of
C. rosa was delayed due to lack of available host fruits.. However, in October 2012,
collection of 1.5 kg of guava fruits from Mwanjamba, Msambweni, Coastal region
yielded 21 adult flies which were utilized for colony initiation. Mwajamba is located
at 040 18’21”S and 0390 29’88”E, 106 m above sea level.
In the highlands, C. rosa have been traditionally reared from mango. In June 2012,
one hundred and two (102) kilograms of mango fruits (variety Van Dyke) were
collected from Kithoka, Meru, Central Province and processed in the laboratory for
fruit flies. The fruits yielded 29 adult C. rosa and the insects were utilized for colony
establishment. Kithoka is located at 00005’59”N, 037040’40”E and 1425 m above sea
level.
Materials and methods
Colony establishment. Due to irregular availability of fruits, attempts were made to
establish colonies of the two populations of C. rosa on solid diet. Eggs of each
population of C. rosa were collected using a guava or mango dome (that had the pulp
and the seed removed) that was placed into fly stock colonies for 1 h. Eggs were
carefully removed from the underside of the dome with a fine camel hair brush and
placed on a 9-cm diameter moist blotting paper. After 36 h, 100 newly emerged larvae
from the above lots of eggs were gently introduced with a fine camel’s hair brush onto
the surface of 100 g of carrot-based diet (Table 1) (Ekesi et al., 2007) in open 150 ml
plastic cups. This cup was nested on a larger 300 ml plastic cup with sterile sand at the
bottom. Mature larvae exited the diet and pupariated in the sand.
3
An experiment consisted of 5 cups of diet containing 100 larvae, with each cup
serving as a replicate. At every generation tested, records were kept on the following
(1) larval stage duration, (2) % puparia recovered from diet, (3) weight of puparia, (4)
adult emergence, (5) fecundity and (6) fertility over a 10-d period. Percent pupal
recovery was calculated based on the initial number of larvae introduced into each
container of rearing medium. Pupal weight was based on four lots of 20 puparia from
each replicate. Adult emergence was based on four lots of 20 puparia from each
replicate that were placed in screened 12 cm diameter plastic containers and observed
for a period of 21 d. Fecundity and fertility were based on daily egg collections from
10 pairs of flies held after a pre-oviposition period of 7 d. Eggs were collected using a
mango or guava dome and hatch rate was assessed after 72 h. In all experiments adults
were fed on a diet consisting of 3 parts sugar and 1 part enzymatic yeast hydrolysate
ultrapure (USB Corporation, Cleveland, OH), and water on pumice granules. All
experiments were carried out in a room maintained at 28  1C, 50  8% RH with a
photoperiod of 12:12 (L:D).
Results and discussion. There were significant differences between the two
populations of C. rosa evaluated for the following quality control parameters: larval
development (F=19.87; 1,40; P=0.0001), % pupal recovery (F=28.90; 1,40;
P=0.0001), adult emergence (F=74.11; df=1,40; P=0.0001) and fertility (F=18.19;
df=1,40; P=0.0001). Pupal weight (F=2.17; df=1,40; P=0.6541) and fecundity
(F=2.13; df=1,40; P=0.6543) did not differ significantly across the two populations.
Significant differences were also observed for the following quality control
parameters over the five generations of C. rosa: larval development (F=78.11; 4,40;
P=0.0001), % pupal recovery (F=76.12; 4,40; P=0.0.0001), adult emergence:
F=81.01; df=4,40; P=0.0989), fecundity (F=52.54; df=4,40; P=0.0001) and fertility
(F=60.22; df=4,40; P=0.0001). Pupal weight (F=1.06; df=4,40; P=0.8865) was not
affected by generation of rearing.
Table 1. Mean larval duration (± SE) and % pupal recovery (± SE) of two
populations of adult C. rosa reared on carrot-based artificial diet
_____________________________________________________________________
Larval developmental period (d)
% Pupal recovery
________________________
___________________
GeneHighland
Lowland
Highland
Lowland
ration
C.rosa
C. rosa
C. rosa
C. rosa
_____________________________________________________________________
P
22.4 ± 0.8a 18.2 ± 0.5b
30.5 ± 1.2a 15.8 ± 1.1b
1
21.8 ± 1.1a 18.7 ± 1.2b
34.6 ± 1.8a 20.2 ± 0.8b
2
22.4 ± 0.6a 18.5 ± 0.1b
50.4 ± 1.5a 30.2 ± 1.8b
3
20.1 ± 0.5a 16.2 ± 1.1b
64.3 ± 1.2a 38.8 ± 2.4b
4
20.2 ± 0.4a 16.8 ± 0.7b
66.4 ± 2.1a 42.6 ± 2.4b
_____________________________________________________________________
For each quality parameter, means within a row followed by the same letter do not
differ significantly by t test (P=0.05).
4
Table 2. Mean pupal weight (± SE) and adult emergence (± SE) of two
populations of C. rosa reared on carrot-based artificial diet
__________________________________________________________________
Pupal weight (mg)
% adult emergence
_____________________
________________________
GeneHighland
Lowland
Highland
Lowland
ration
C. rosa
C. rosa
C. rosa
C. rosa
___________________________________________________________________
P
8.8 ± 1.1a
9.1 ± 0.5a
70.6 ± 2.1a
46.2 ± 1.8b
1
8.7 ± 0.6a
8.6 ± 1.1a
72.2 ± 1.4a
50.2 ± 1.5b
2
10.3 ± 1.1a 10.6 ± 1.2a
72.6 ± 2.5a
54.6 ± 4.6b
3
12.2 ± 1.5a 11.9 ± 1.6a
74.2 ± 3.8a
56.2 ± 2.8b
4
12.4 ± 1.2a 12.2 ± 1.8a
78.2 ± 1.8a
50.8 ± 2.8b
____________________________________________________________________
For each quality parameter, means within a row followed by the same letter do not
differ significantly by t test (P=0.05).
Table 3. Mean fecundity (± SE) and fertility (± SE) of two populations of C. rosa
reared on carrot-based artificial diet
___________________________________________________________________
Fecundity (10-day)
Fertility, % egg hatch
____________________
________________________
Gen eHighland
Lowland
Highland
Lowland
ration
C.rosa
C. rosa
C. rosa
C. rosa
____________________________________________________________________
P
78.8 ± 10.4a 80.2 ± 8.6a
50.4 ± 1.2b
36.2 ± 0.8b
1
74.2 ± 12.6a 78.4 ± 14.4a
54.4 ± 2.6b
34.5 ± 2.6b
2
98.2 ± 21.1a 104.5 ± 18.5a
62.1.0 ± 2.4b 44.2 ± 4.1b
3
102.5 ± 27.4a 104.2 ± 17.4a
68.4 ± 1.8a
40.8 ± 1.8b
4
154.2 ± 18.6a 149.5 ± 24.2a
70.2 ± 1.8a
48.9 ± 3.6b
_____________________________________________________________________
For each quality parameter, means within a row followed by the same letter do not
differ significantly by t test (P=0.05).
Across generations, larval duration of the highland population were longer (20.1-22.4
d) compared with the lowland population (16.2-18.7 d) (Table 1). Similarly, across
generations, % pupal recovery of the highland population was higher (30.5 to 66.4%)
compared to the lowland population (15.8 to 42.6%) (Table 1). In both populations,
pupal recovery increased from parent generation to the fourth generation.
Pupal weight was not affected by population or generation but became heavier with
increasing generations (Table 2). Significantly higher adults emerged from the
highland population (70.6 to 78.2%) compared with the lowland population (46.2 to
56.2%) (Table 2).
Ten day fecundity was not affected by population but increased with increasing
generations of rearing (Table 3). Across generations, egg fertility in the highland
population was significantly higher (50.4 to 70.2%) than that of the lowland
population (34.5 to 48.9) (Table 3).
5
Both populations of C. rosa can be successfully reared on artificial carrot-based diet
but the highland population appears to be easily amenable to artificial rearing than the
lowland population. Generally, laboratory colonization and mass production of fruit
flies on artificial diet may require several generations for the insects to adapt to the
artificial diet (Kamikado et al. 1987, Souza et al. 1988, Economopoulos 1992). It is
therefore not surprising that quality control parameters of the highland population
differ from that of the highland population. For example, Economopoulos (1992)
showed that even after nine generations, fecundity of wild C. capitata did not match
the levels of laboratory-adapted flies. Clearly, the lowland C. rosa population require
a longer period to adapt to the medium but long term monitoring is required to see
how long it will take for this population to reach the plateau of the highland
population.
2. Assess the effect of temperature on development and survival of lowland
and highland populations of C. rosa.
Background and rationale. In Kenya, Ceratitis rosa has been observed to split up
into two different groups (the lowland and highland populations) which can be
differentiated genetically as well as morphologically (at least in the males). It is
probable that they might also both have different physiological patterns with one
entity being more cold tolerant than the other and with the potential for varying
invasive powers. In this regard we propose to establish colonies of the two populations
of C. rosa from known host plants of the insect in Kenya (Copeland et al., 2006).
Colony establishment will follow the same methodology as previously described. The
different populations will be shared with participants in the CRP as required.
Materials and methods
Insect culture. Two C. rosa populations were used for this experiment and initial
stock culture of highland population originated from infested mango fruits collected at
a smallholder farm in Meru and the larvae were subsequently reared on a carrot-based
artificial diet (hereafter referred to as diet) in the laboratory. The lowland population
originated from fallen guava fruits collected from a smallholder farm in Mwamjamba,
Kenya and also subsequently reared on carrot-based diet. Both colonies were
maintained for 5 generations on the artificial diet at the Animal Rearing and
Containment Unit (ARCU) of the International Centre of Insect Physiology and
Ecology (icipe), Nairobi, Kenya before commencement of the experiments. Rearing
conditions are maintained at 28 ± 10C, 50  8% RH and photoperiod of L12: D12.
Egg collection. Eggs of C. rosa were collected from the stock colony by offering to
mature female flies, ripe mango or guava dome (fruit skin that has the seed and pulp
scooped out). The domes were placed over a 9 cm diameter Petri dish lined with
moistened filter paper. Domes were maintained in 30 x 30 x 30 cm perspex cage at 28
± 10C , 50  8% RH. Each dome was pierced with an entomological pin (38 mm long,
0.3mm diameter) to facilitate oviposition. Eggs were collected within 1 h of
oviposition using a moistened fine camel’s hair brush.
Effect of temperature on development and survival of C. rosa. Egg stage: 100 eggs
were counted and carefully lined on a rectangular piece of moistened sterilized black
6
cloth in a Petri dish. The Petri dishes were immediately transferred to thermostatically
controlled environmental chambers (MLR-153, Sanyo, Japan) set at 5 constant
temperatures of 10, 15, 20, 25, 30, 33 and 350C (± 10C) and 50  8% RH, 12:12 L:D
photoperiod. Duration of egg stage was observed at 6-hourly intervals under a
binocular microscope for determination of egg hatch.
Larval stage: 100 first instar larvae (~1 h old) of each population were counted and
carefully transferred on a cellulose sponge in a Petri dish and thereafter placed on top
of a 50 g of diet on a 5 x 5 x 3 cm plastic container. Twenty four hours after the larvae
have settled in the diet, the tray was transferred in to larger rectangular plastic rearing
containers (7 x 7 x 5 cm) carrying a thin layer (~ 0.5 cm) of moist sterilized sand at
the bottom for pupation and into the environmental chambers. The top of the plastic
containers were screened with light cloth netting material for ventilation. The
containers were then maintained at the same constant temperature in the
environmental chambers. Normally, mature late third instar larvae leave the Petri
dishes containing the artificial diets ad libitum and jump into the sand in the larger
containers to pupate. Starting at 5 days after the larvae were exposed to artificial, the
number of puparia in the sand was recorded daily by sifting and record of larval
duration were kept for each population at each temperature regime.
Pupal stage: One hundred newly formed pupae (~ 1 h old) were obtained from the
culture and held in small-ventilated transparent cylindrical plastic cages (5.5 x 12.5
cm). The pupae were transferred to the same 7 constant temperatures and records were
kept of duration to adult eclosion.
Statistical analysis
General analysis: To examine the effects of temperature on life history parameters,
data for developmental time and survivorship was subjected to two-way analysis of
variance (ANOVA). Log10 ± 0.5 and arcsine square root transformation were used
respectively, on counts and percentages before statistical analyses (Sokal and Rohlf,
1981). When treatment effects were significant, means were separated using StudentNewman-Keul’s (SNK) test.
Linear model: The linear portion of the developmental rate curve [R(T)=a + bT] was
modeled using regression analysis where T was temperature, and a and b were
estimates of the intercept and slope, respectively. The lower temperature threshold
(Tmin) was estimated by the intersection of the regression line at R(T) = 0, T0 = -a/b.
Degree-day (DD) requirements (thermal constant, K) was calculated using the inverse
slope of the fitted linear regression line (Campbell et al. 1974).
Nonlinear model: The nonlinear relationship between developmental rate r(T) and
temperature T was fitted to the Brière model, which allows the estimation of the
upper and lower developmental thresholds (Brière et al. 1999). Brière-1 model was
used and described as:
1/D=a x T x (T – T0) x Sqrt(TL – T)
Where T0 (tmin) is the lower threshold, TL (tmax) the lethal temperature is the upper
threshold and a is an empirical constant.
7
The following statistical items were used to assess the goodness-of-fit: the coefficient
of determination (for linear model; R2) or the coefficient of nonlinear regression (for
nonlinear models; R2) and the residual sum of squares (RSS). Higher values of R2 and
lower values for RSS reveal a better fit. For the linear regression, the data points at
350C which deviated from the straight line through the other points were rejected for
correct calculation of regression (Campbell et al. 1974). The model fitting was
implemented in R 2.13.1 (R Development Core Team, 2011) using the nls function.
Results and discussion
Effect of temperature on stage development. The time required for eggs to hatch
ranged from 7.83 days at 150C and decreased to 1.75 days at 330C (F = 29.1.2, d.f = 4,
15 P=0.0001) in the highland population. In the lowland population, egg development
was also longest (7.54 days) at 150C and shortest (1.63 days) at 330C (F=42.98;
df=5,18; P=0.0001) (Table 4). At 350C, eggs of the highland population of C. rosa,
failed to hatch. However, egg developed at 350C in the lowland population and took
1.75 days to hatch. In both populations, eggs did not develop at 100C.
The linear regression model showed a strong positive linear relationship between
temperature and egg development rate for highland (R2 =0.98) and lowland (0.94)
populations (Fig 1) with a lower development threshold of 10.60C and 10.10C for
highland and lowland populations, respectively. The egg stage required 38 degreedays (DD) to complete development in the highland population and 34 DD in the
lowland population. For the highland population, the parameter estimates for the
Brière-1 nonlinear model predicted the lower temperature threshold of 11.10C and the
upper temperature threshold of 34.80C. The rate of development increased with
temperature until the curve reached an optimum and then decreased rapidly as
temperatures reached the upper temperature threshold (Fig. 1).
At larval stage, the trend was similar to the egg stage with development periods
decreasing from 24.91 days at 150C to 9.50 days at 350C (F = 56.63, d.f = 4, 15 P=
0.0001) for highland population; and 22.9 at 150C to 7.69 at 350C (F = 113.96, d.f = 5,
18 P= 0.0001) in the lowland population (Table 4). At 350C, larvae did not develop in
the highland population. Also at 100C, no development occurred in the both
populations (Table 4). At 300C, the lowland population developed faster than the
highland population (Table 4).
The linear regression between temperature and development rate for this stage was
positive for the highland population (R2 = 0.94) as well as the lowland population
(R2=0.97) (Fig. 1). Highland C. rosa required 203 DD above the development
threshold of 10.10C to complete development from larval stage to the pupal stage. The
lowland population took 177 DD to develop above a threshold of 10.30C. Parameter
estimates for the Brière-1 nonlinear model predicted the lower and upper temperature
threshold of 10.40C and 35.00C for the highland population. For the lowland
population, Brière-1 nonlinear model estimated lower and upper developmental
threshold of 10.60C and 40.80C, respectively.
In the highland and lowland populations, temperature had a significant effect on
development of pupae: (F =149.45, d.f = 3, 12 P< 0.0001) and (F = 385.43, d.f = 3, 12
P= 0.0001), respectively. In both populations, the longest duration occurred at 150C:
8
highland =35.42 days and lowland = 34.25 days (Table 4). There was no eclosion at
10, 33 and 350C in both populations (Table 4).
The linear regression between temperature and development rate for this stage was
positive for both populations: Highland - R2 = 0.98 and lowland - R2 = 0.96 with a
lower development threshold of 9.6 and 10.00C, respectively (Fig.1). The puparium
required 228 and 182 DD to complete development in the highland and lowland
populations, respectively. Parameter estimates for the Brière-1 nonlinear model
predicted the lower and upper temperature threshold of 9.980C and 35.00C for the
highland population. For the lowland population, Brière-1 nonlinear model estimated
lower and upper developmental threshold of 10.10C and 35.00C, respectively.
In both populations, total developmental duration was longest at 150C (Highland
=68.16 days; Lowland=64.69 days) and shortest at 300C (Highland=22.64 days;
Lowland=19.76 days) (Table 4).
Survival rates. At egg stage, survival ranged between 43.5% at 330C to 63.5% at 250C
(F = 4.96, d.f = 4, 15 P = 0.0095) in the highland population and 72.0% at 350C to
91.5% at 250C in the lowland population (F = 5.42, d.f = 5, 18; P = 0.0033) (Table 5).
In the highland population, survivorship at larval stage ranged between 35.75% at
330C to 77.00 % at 250C (F = 14.36, d.f = 4, 15 P= 0.0001). In the lowland
population, survival rate was lowest (48.25%) and highest (89.25%) at 250C (F =
24.24, d.f = 5, 18 P= 0.0001) (Table 5).
During the pupal stage, survival ranged from 61.25% at 300C to 94% at 250C in the
highland population (F = 22.76, d.f = 3, 12 P= 0.0001). In the lowland population,
survival ranged from 75.5% at 300C to 95.5% at 250C (Table 5).
The ranges in duration of immature stages are in general agreement with those
reported for other Ceratitis species (Delrio et al., 1986; Fletcher, 1989; Vargas et al.,
1996; Duyck and Quilici, 2002). Duyck and Quilici (2002) published the first report
on development of C. rosa at temperatures of 15-350C. The authors reported total
development in days of 18.8–65.7 days. In our study, total development of the
highland population of C. rosa took 22.6-68.2 days while lowland population took
19.8-64.7 days. Results from the lowland population therefore closely mirrored those
reported for populations of C. rosa from La Reunion. Values for the temperature
threshold and thermal constant were also consistent with previous studies. Duyck and
Quilici (2002) reported lower developmental thresholds for the egg, larval and pupal
stages as 9.8, 3.1, and 11.0, respectively. Apart from the larval stage, the estimated
thresholds from our study are generally in agreement with these authors for both
populations. No previous studies are available in literature with regard to upper
developmental threshold for C. rosa. Physiologically, Brière-1 nonlinear model
predicted that both populations seem to have similar lower temperature thresholds but
vary in their tolerance to higher temperatures. In our study, these values ranged from
35.0-40.80C across the developmental stages in the lowland population but was
exactly at 35.00C for the highland population. Clearly, the lowland population
tolerates higher temperatures than the highland population.
9
Table 4. Mean ± SE developmental time (d) of immature stages of highland and coastal populations of Ceratitis rosa at seven constant temperatures
_________________________________________________________________________________________________________________________________________________________
Temperature
(°C)
Egg
Larva
Pupa
Total (days)
____________________________
______________________________
_____________________________
______________________________
Highland
Coast
Highland
Coast
Highland
Coast
Highland
Coast
_________________________________________________________________________________________________________________________________________________________
10
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
No emergence
No emergence
-
-
15
7.83 ± 0.64aA
7.54 ± 0.40aA
24.91 ± 1.05aA
22.90 ± 0.94aA
35.42 ± 0.95aA
34.25 ± 0.90aA
68.16 ± 2.17aA
64.69 ± 1.81aA
20
3.75 ± 0.48bA
3.17 ± 0.47bA
14.25 ± 0.30bA
13.54 ± 0.53bA
22.38 ± 0.48bA
19.63 ± 0.40bB
40.38 ± 0.85bB
36.33 ± 0.89bA
25
2.63 ± 0.29cbA
2.25 ± 0.30bcA
11.88 ± 1.29bcA
9.88 ± 0.35cA
15.75 ± 0.44cA
11.88 ± 0.35cB
30.25 ± 1.11cA
24.01 ± 0.82cB
30
1.88 ± 0.35cA
1.75 ± 0.23cA
9.88 ± 0.22cA
8.88 ± 0.12cdB
10.88 ± 0.55dA
9.13 ± 0.22dA
22.64 ± 1.26dA
19.76 ± 0.48dA
33
1.75 ± 0.30cA
1.63 ± 0.12cA
9.50 ± 0.38cA
8.25 ± 0.44cdA
No emergence
No emergence
-
-
35
0.00 ± 0.00B
1.75 ± 0.40cA
0.00 ± 0.00B
7.69 ± 0.17dA
No emergence
No emergence
-
-
_________________________________________________________________________________________________________________________________________________________
Means in the same column followed by the same upper case and in the same row followed by the same lower case letter are not significantly different [ANOVA and Student
– Newman – Keul’s (SNK) test, P < 0.05].
10
Table 5. Mean ± SE survivorship (%) of immature stages of highland and coastal populations of Ceratitis rosa at seven constant temperatures
______________________________________________________________________________________________________________________________
Temperature
(°C)
Egg
____________________________
Highland
Coast
Larva
______________________________
Highland
Coast
Pupa
_____________________________
Highland
Coast
___________________________________________________________________________________________________________________________________________
10
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
15
51.0 ± 3.09abB
84.50 ± 2.15abA
54.75 ± 2.84bB
70.00 ± 1.81dA
73.75 ± 3.28cA
80.25 ± 1.87cA
20
54.0 ± 4.68abB
81.80 ± 2.66abA
62.25 ± 2.94bB
80.00 ± 3.91bcA
86.25 ± 3.15bA
88.25 ± 1.38bA
25
63.50 ± 1.95aB
91.50 ± 2.76aA
77.00 ± 4.61aB
89.25 ± 1.53aA
94.00 ± 1.73aA
95.5 ± 1.22aA
30
62.30 ± 2.22aB
90.50 ± 1.71aA
69.75 ± 4.43abB
83.25 ± 1.79abA
61.25 ± 2.01dB
75.5 ± 2.63cA
33
43.50 ± 4.35bB
91.00 ± 2.65aA
35.75 ± 3.10cB
72.75 ± 1.22cdA
0.00 ± 0.00
0.00 ± 0.00
35
0.00 ± 0.00B
72.00 ± 4.58bA
0.00 ± 0.00B
48.25 ± 3.65eA
0.00 ± 0.00
0.00 ± 0.00
______________________________________________________________________________________________________________________________
Means in the same column followed by the same upper case and in the same row followed by the same lower case letter are not significantly different
[ANOVA and Student – Newman – Keul’s (SNK) test, P < 0.05].
11
Fig. 2. Effect of constant temperature on development rates (1/ duration in days) of different life stages of highland and coastal populations of Ceratitis rosa:
egg, larva and pupa.
12
3. Assess mating compatibility between lowland and highland populations of
C. rosa.
Background: Because of its economic importance, area-wide sterile insect technique
(SIT) has been proposed for the management of C. rosa and there is mounting interest
for the application of the technique for management of the pest in Africa. The
potential existence of cryptic species complex may pose challenges to area-wide SIT,
especially if reproductively isolated morphotypes exist because releases of sterile
males of the wrong population will not induce sterility into the target population. To
overcome this problem, studies of mating compatibility is essential.
Materials and methods
Source of insects. Both populations of C. rosa originated from guava and mango
fruits as earlier described. Ceratitis fasciventris originated from coffee berries
collected from Ruiru, Nairobi.
Compatibility test. The test followed the standard procedures to evaluate mating
compatibility as outlined in the FAO/IAEA/USDA Product Quality Control Manual
(2003). Two to 3 days before adult emergence, pupae from all three populations were
placed in 15 cm diameter x 45 cm high cylindrical Plexiglass cages. At emergence,
adults were sorted by sex and placed in similar cages containing water on pumice
granules and 3:1 parts of sugar and enzymatic yeast hydrolysate as food. One to 2
days before reaching sexual maturity (10 to 14 days) males and females of each
population were marked on the thorax with a small dot of water-based paint. Thirty
marked males and 30 marked females of each population were placed in a 30 x 30 x
30 plexiglas cage with water and food. The tests were carried out in 5 x 5 x 5 m field
cage fitted with red lights. In each cage, three potted mango trees, 1.5 m in height with
~ 1.5 m dia. Canopy provided flies with arena for mating and resting. At 19:30 h on
the day of each test, marked virgin males of two different populations were released
inside a 5 x 5 x 5 m cage and 15 min later, virgin females from each of the same two
populations were added. Two observers in each cage recovered mating couples from
the tree and cage walls and ceiling recording each time: colour (origin) of male and
female, time at which copulation initiated, and mating location. Soon after the
detection of a mating pair, the couple was gently captured in a small (4 cm in
diameter, 4cm high) plastic cup which was capped and placed over a plastic tray to
record the time at which copulations ended. Latency to mate was based on the time
from the release of the females to the beginning of a given copulation. Copulation
duration was estimated as the time the couple disengaged minus the time they started
to copulate. Flies were observed for ca. 3 hours, a time lapse that guarantees the total
period of sexual activity for populations of all the insects (S. Ekesi et al., unpublished
data). Thereafter mated couples and remaining unmated adults were taken to the
laboratory. Given the difficulty in establishing the coast population of C. rosa, only 3
replications have been conducted and results presented should still be considered as
preliminary.
Results and discussion: The percentage of the different populations copulating and
indices of sexual compatibility are presented in Tables 6 and 7. The results obtained
showed a high degree of mating incompatibility between the 2 populations of C. rosa
and also between the 2 populations and C. fasciventris. The ISI values ranged from
13
0.75 to 0.90 with the Highland C. rosa x the Lowland C. rosa showing the highest
degree of isolation. All matings were achieved by males of the Lowland C. rosa
population. No significant difference was observed in latency to mate among the
mating combinations: Highland C. rosa x Lowland C. rosa (F=1.76; df=2,6;
P=0.4004), Highland C. rosa x C. fasciventris (F=2.56; df=2,6; P=0.6540) and
Lowland C. rosa x C. fasciventris (F=1.98; df=2,6; P=0.7115) (Table 8). Mating
duration was also similar for all mating combinations: Highland C. rosa x Lowland
C. rosa (F=3.10; df=2,6; P=0.1701), Highland C. rosa x C. fasciventris (F=1.43;
df=2,6; P=0.6021) and Lowland C. rosa x C. fasciventris (F=2.76; df=2,6; P=0.5013)
(Table 8). Generally, over 80% of the mating occurred on the tree canopy for all the
combinations. Overall, this preliminary observation provides some indication of the
existence of variability among populations of C. rosa in Kenya. The relatively high
levels of isolation between the 2 populations suggest the possible existence of
different taxonomic entity. However, having conducted only 3 replications, it is
premature to conclude that the highland populations of C. rosa are completely distinct
from the lowland populations. More replications coupled with molecular studies are
required to confirm the results presented here.
Table 6. Sexual compatibility of two Ceratitis rosa populations and C. fasciventris
in pairwise combinations
_____________________________________________________________________
No. of couples
_______________________________________________
Populations tested
AA
AB
BA
BB
_____________________________________________________________________
Highland C. rosa + Lowland C. rosa 16.3±1.2 1.0±1.0 1.0±1.0 20.0±1.4
Highland C. rosa + C. fasciventris
15.0±2.1 2.7±1.2 1.0±1.0 16.0±0.5
Lowland C. rosa + C. fasciventris
20.3±3.8 1.7±0.3 1.7±0.4 14.3±1.8
_____________________________________________________________________
Table 7. Performance indices in sexual compatibility test between two Ceratitis
rosa populations and C. fasciventris
_____________________________________________________________________
Populations tested
PM
ISI
MRPI
FRPI
_____________________________________________________________________
Highland C. rosa + Lowland C. rosa 63.8±2.2 0.90±0.05 -0.10±0.03 -0.10±0.02
Highland C. rosa + C. fasciventris
57.8±1.8 0.75±0.01 0.02±0.01 -0.08±0.01
Lowland C. rosa + C. fasciventris
63.3±3.4 0.84±0.06 0.17±0.06 00.0±0.00
_____________________________________________________________________
Highland C. rosa (HLR) + Lowland C. rosa (LLR) (AA = HLR♂ + HLR♀; AB =
HLR♂ + LLR♀; BA = LLR♂ + HLR♀; BB = LLR♀ + LLR♂); Highland C. rosa +
C. fasciventris (AA = HLR♀ + HLR♂; AB = HLR♂ + CF♀; BA = CF♂ + HLR♀;
BB = CF♀ + CF♂); Lowland C. rosa + C. fasciventris (AA = LLR♀ + LLR ♂; AB =
LLR♂ + CF♀; BA = LLR♀; BB = CF♀ + CF♂).
14
Table 8. Latency to mate and copula duration (mean ± SE) for heterotypic and homotypic crosses of 2 populations of C. rosa
and 1 of C. fasciventris
__________________________________________________________________________________________________________
Mating
Mating combination
Latency to mate
Copula duration
Combination
(Male – Female)
(Minutes)
(Minutes)
__________________________________________________________________________________________________________
Highland C. rosa-Lowland C. rosa Highland – Highland
13.34 ± 2.11
332.4 ± 11.8
Highland - Lowland
15.78 ± 5.16
302.7 ± 23.5
Lowland – Highland
21.46 ± 3.50
365.2 ± 18.2
Lowland – Lowland
18.76 ± 2.54
311.9 ± 30.1
Highland C. rosa – C. fasciventris
Highland – Highland
11.55 ± 4.22
315.6 ± 41.7
Highland – C. fasciventris
21.13 ± 4.71
365.2 ± 18.2
C. fasciventris – Highland
17.17 ±5.14
333.8 ± 22.6
C. fasciventris – C. fasciventris
26.11 ± 4.76
373.2 ± 25.5
Lowland C. rosa – C. fasciventris
Lowland – Lowland
20.42 ± 3.50
365.2 ± 18.2
Lowland – C. fasciventris
17.67 ± 2.65
309.4 ± 16.9
C. fasciventris – Lowland
12.17 ±4.11
343.1 ± 30.8
C. fasciventris – C. fasciventris
16.22 ± 3.76
311.4 ± 17.5
__________________________________________________________________________________________________________
15
Bactrocera cucurbitae
1. Catalogue B. cucurbitae host plants and carry out host preference studies,
preferably under field cage conditions.
Background: Previously documented species of Bactrocera in Kenya include melon
fly Bactrocera cucurbitae Coquillett, the Sri Lankan fruit fly, B. invadens, olive fruit
fly B. oleae (Gmelin), B. biguttula (Bezzi) and B. munroi White (White and ElsonHarris 1992; Copeland et al. 2004; Ekesi et al., 2006). Among the five species, B.
invadens and B. cucurbitae are believed to the most destructive to fruits and
vegetables (Ekesi and Billah, 2007). Most frugivorous tephritids within the genus
Bactrocera are known to attack a wide range of fruit, vegetable and wild plant species.
For example, B. invadens have been reported from over 40 host plants ( Vayssières et
al. 2005; Mwatawala et al. 2006; Rwomushana et al., Georgen et al., 2011). The host
range of B. cucurbitae are believed to be primarily cucurbits (White and Elson-Harris
1992), but there are no published information on the diversity of plant species attacked
by the insect in Kenya and the knowledge of it preferred host plant is also lacking.
Because of the growing importance of B. cucurbitae there is the need to document the
host plants of this important quarantine pest. The main objective of this study,
therefore, was to catalog the host plants of B. cucurbitae in Kenya given its
importance as a major quarantine pest in order to provide necessary information that
may be useful for management of the pest. We also conducted host preference studies
in field cages that included 9 of the major host and export fruits and vegetables that
were infested in the field survey to ascertain the most preferred host plant of the
insect.
Materials and methods
Field surveys, fruit collection, handling and processing. Host fruit survey was
carried out from April 2012 to May 2013 2006, in three provinces (Table 9) in Kenya
where B. cucurbitae had been previously confirmed with Cue lure baited traps (Ekesi
et al. unpublished data). In Each province, one location with large diversity of fruits
was selected for fruit sampling. At the Coast Province, surveys were concentrated at
Taveta covering the fruit and vegetable irrigation scheme at the border with Tanzania.
At the Eastern Province, sampling was done at Nthagaiya division covering part of
Karurumo and Kigumo. In Central Province, sampling location was concentrated
largely at Muranga division.
Fruits were collected from cultivated fields, backyard gardens, bushlands and
protected reserves. Fruit samples collected included mature green to ready-to-harvest
and those with visible symptoms of fruit fly damage both from the plant and from the
ground. Fruit collections of the different plant species were separately placed in
perforated polyethylene bags in the field for transport to the rearing facility. At the
rearing facility, fruits were counted, weighed and secured in well-aerated rectangular
plastic containers and processed according to methodology described by Copeland et
al. (2007). Fruits were held at ambient conditions for 4-6 weeks depending on the fruit
species. Rearing cages were checked daily and puparia were picked from the sand
with a pair of soft forceps, counted and placed in petri dishes with moistened filter
16
paper. Emerging tephritids were provided with an artificial diet that consisted of a
volumetric mixture of 1:3 enzymatic yeast hydrolysate and sugar, and water was
provided in pumice granules. Flies were allowed to feed for 4 days until full adult
development and body colorations were attained. They were then killed by placing
them in a freezer, and later preserved in 70% alcohol. All specimens were shipped to
the icipe Biosystematics unit for identification where a reference collection is kept.
Samples of flower, fruit, leaf and/or twig from unknown plant species were also
collected, pressed and bagged. The collected plant samples were identified using the
keys of Kenya trees, shrubs and lianas (Beenjte, 1994).
Results and discussion: Bactrocera cucurbitae was reared from a total collection of
17 plant species comprising 10 families covering Cucurbitaceae, Solanaceae,
Anarcadiaceae, Rutaceae and Myrtaceae from surveys carried out at the Coast,
Eastern and Central Provinces of Kenya (Table 9). Across the localities, highest
infestation was recorded from Momordica charantia (9.8 to 16.2flies/kg fruits),
Citrullus lanatus (8.5 to 11.2 flies/kg fruits) and Lycopersicum esculentum (5.7 to 12.1
flies/kg fruits) (Table 9). The next most important group of fruits included Cucumis
sativus, Cucurbita maxima and C. moschaata with infestation indices ranging from
2.6 to 4.4 flies/kg fruits. In general, there was a tendency for higher fruit infestation in
Taveta (lowland) than in Muranga and Nthagaiya (highland) (Table 9). Bactrocera
cucurbitae was also recorded from non-traditional host plants such as Mangifera
indica, citrus sinensis, C. reticulata and Psidium guajava but infestation did not
exceed 0.5 flies/kg fruits (Table 9). Vayssières et al (2007) reported that although B.
cucurbitae largely infested plant species from the family Cucurbitaeae, the pest was
also recorded from cashew, mango, citrus and carambola. Frequently and in majority
of the host plants, B. cucurbitae shared host with B. invadens and in some highly
preferred host plants like C. moschata, B. invadens predominated. Generally, all the
plants species listed here have been reported as host to B. cucurbitae. However, L.
esculentum require careful attention because infestation in this important cultivated
host plants compared highly with other traditional cucurbitaceous host plants. The
reason for this switch is not very clear but warrant further investigation.
2. Establish Bactrocera cucurbitae colonies from different hosts and habitats
Colonies of two populations of B. cucurbitae (from tomato and bitter gourd) have
been established using the whole fruit rearing technique and vibrant colonies are
available for research and shipment to partners upon request.
3. Field cage host plant preference studies
Materials and methods
The experiments were conducted in 5 x5 x 5 m field cage located at the icipe field
station at Mbita Point using two populations of B. cucurbitae: (1) those reared from
bitter gourd and (2) those reared from tomato. Three fruits species (bitter gourd,
tomato, water melon) that ranked high in infestation indices from the field collections,
three (pumpkin, cucumber, butternut) that were rated as moderately infested and
another three (pepper, guava, mango) with low infestation indices were selected for
host preference studies. Each experimental cage was divided equally into 9 subunits
and each unit held one fruit species supported by a string from the roof of the cage.
17
All fruit species were tested when they were at harvest maturity. Three hundred adult
B. cucurbitae (consisting of 150 females and 150 males) at 2-3 weeks old were then
released inside the cages for a period of 24 hrs. Flies were fed on 1:3 volumetric
mixture of enzymatic yeast hydrolysate and sugar in petri dishes. Water was also
provided on pumice granules. After 24 h, all fruit species were removed and incubated
individually as described for field surveys. Records were kept for pupal recovery and
percentage of adult emergence from the total puparia recovered. Four replicated cages
were maintained for each population of B. cucurbitae.
Results and discussion. In the experiment in which bitter gourd populations of B.
cucurbitae were used, significantly higher puparia were recovered from M. charantia
(134.2) compared to the other host fruit species (F=45.12; df=8,24; P=0.0001) (Fig.
3A). The next preferred lot of fruits were C. lanatus, C. sativus, C. maxima and C.
mosschata with infestation ranging from 74.3-76.5 (Fig. 3A). In experiment involving
the tomato population of B. cucurbitae, the insect exclusively preferred tomato (117.2
puparia) compared to the other host plants (10.6-18.4) (F=76.15; df=8,24; P=0.0001)
(Fig. 3B). In this particular case, no puparia were recovered from guava and mango
(Fig. 3B). In both populations of B. cucurbitae and on all host plants, adult emergence
ranged from 78-82% and did not differ significantly across treatments: bitter gourd
population (F=1.21; df=8,24; P=0.4023) and tomato population (F=1.14; df=8,24;
P=0.4012) (Fig. 3B). We conclude that populations of B. cucurbitae vary in their
preference to host plants and this preference is driven also by variation among
population of the pest. Although outbreak of population of B. cucurbitae on tomato is
common, there is the need for a closer look at the populations attacking tomato at the
molecular level to ascertain their species identity.
Shipment of materials to partners
During the period under review fruit fly specimens (specifically C. rosa) of different
quantities were shipped to the following partners for difFrrent activities related to the
CRP:
1. Dr. Gary Steck – USA
2. Dr. Marc De Meyer - Belgium
3. Dr. Lucie Vanickova – Czech Republic
18
Table 9. Fruit infestation indices for Bactrocera cucurbitae in fruit samples collected from February 2012 to May 2013 in Kenya
________________________________________________________________________________________________________________
No. flies/kg fruitb
Cucurbit
No. of
Fruit wt.
% fruit
_______________
Location
species
fruits
(kg)
infested
Bc
Bi
________________________________________________________________________________________________________________
Taveta
Momordica charantia L(bitter gourd)
132
29.7
64.3
16.2
3.3
Cucumis sativus L (Cucumber)
125
31.3
27.6
3.7
5.2
Cucumis melo L (Melon)
113
14.2
7.5
1.5
0.0
Cucurbita maxima Duch (Pumpkin)
48
100.5
24.8
4.2
3.8
Cucurbita pepo L. (Zucchini)
102
30.7
12.2
1.3
3.7
Cucurbita moschata (Butter nut)
134
12.5
10.4
3.1
10.4
Citrullus lanatus (T.) Mats (water melon)
51
119.4
58.4
11.3
1.8
Lagenaria siceraria (M.) Standl (Calabash)
111
60.2
7.8
1.5
0.0
Luffa cylindrica (L.) Roen (Luffa)
142
21.7
6.4
1.3
1.4
C. melo var. conomon (Sweet melon)
102
66.2
3.2
0.8
0.0
Capsicum frutescens L. (Pepper)
789
1.3
4.4
0.6
2.6
Lycopersicum esculentum (Tomato)
131
7.7
56.6
12.1
1.4
Solanum melongena (Egg plant)
76
3.5
2.1
1.1
2.8
Mangifera indica (Mango)
109
31.6
8.2
0.5
121.4
Citrus sinensis (Sweet orange)
93
7.4
2.5
0.5
2.1
Citrus reticulata Blanco (Tangerine)
40
2.8
1.5
0.2
1.1
Psidium guajava (Guava)
42
3.8
2.2
0.4
14.7
Muranga
Momordica charantia L(Bitter gourd)
Cucumis sativus L (Ccumber)
Cucurbita maxima Duch (Pumpkin)
Cucurbita pepo (Zucchini)
105
131
21
171
18.9
34.9
52.8
52.1
58.2
11.7
13.8
32.2
10.2
3.1
4.4
1.8
1.8
0.0
1.2
0.0
19
Nthagaiya
Cucurbita moschata (Butter nut)
Citrullus lanatus (T.) Mats (Water melon)
Capsicum frutescens L. (Pepper)
Lycopersicum esculentum (Tomato)
Solanum melongena (Egg plant)
Mangifera indica (Mango)
Citrus sinensis (Sweet orange)
Psidium guajava (Guava)
111
60
601
123
51.9
78
50
58
10.1
125.0
0.8
6.4
2.2
22.1
5.5
4.4
9.1
48.5
10.2
40.1
4.2
4.8
2.1
1.7
3.6
10.2
1.1
6.5
1.8
0.0
0.5
0.2
12.5
1.1
3.4
0.0
0.0
101.2
3.2
11.4
Momordica charantia L(Bitter gourd)
Cucumis sativus L (Cucumber)
Cucurbita pepo (Zucchini)
Cucurbita moschata (Butter nut)
Citrullus lanatus (T.) Mats (water melon)
Capsicum frutescens L. (Pepper)
Lycopersicum esculentum (Tomato)
Mangifera indica (Mango)
Citrus sinensis (Sweet orange)
Citrus reticulata Blanco (Tangerine)
Psidium guajava (Guava)
121
77
160
98
71
923
101
82
66
53
62
22.3
24.8
44.8
11.8
138.2
1.5
5.1
23.5
6.1
3.1
4.1
44.8
12.5
24.2
11.3
38.2
15.3
34.2
3.2
4.2
0.0
2.2
9.8
3.2
1.4
2.6
8.5
1.1
5.7
0.4
0.0
0.0
0.4
0.0
0.0
1.5
10.5
0.0
4.3
1.1
78.9
1.1
2.1
10.6
_________________________________________________________________________________________________________________
Bc = Bactrocera cucurbitae; Bi = Bactrocera invadens
20
180
No. puparia recovered
160
140
120
100
80
60
40
20
0
A
140
No. puparia recovered
120
100
80
60
40
20
0
B
Fig. 3. Host preference and fruit infestation by two populations of B. cucurbitae
in field cages. A = bittergourd population; B = tomato population
21
Activities proposed for the next reporting period
1. The temperature studies will be repeated to validate the results obtained so far.
2. Establish temperature dependent life-table parameters for the two populations
of C. rosa and develop phenological models from the life-table statistics to
predict species risk under different climate change scenarios.
3. Complete mating compatibility studies
4. Others activities as may be suggested and agreed upon during the Tucuman
meeting.