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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

icipeP. O. Box 30772, 00100 GPO, Nairobi, Kenya

Chief Scientific Investigator: Dr. S. Ekesi

June 28, 2013

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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 methodsColony 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.

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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 1C, 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________________________ ___________________

Gene- Highland Lowland Highland Lowlandration C.rosa C. rosa C. rosa C. rosa_____________________________________________________________________P 22.4 ± 0.8a 18.2 ± 0.5b 30.5 ± 1.2a 15.8 ± 1.1b1 21.8 ± 1.1a 18.7 ± 1.2b 34.6 ± 1.8a 20.2 ± 0.8b2 22.4 ± 0.6a 18.5 ± 0.1b 50.4 ± 1.5a 30.2 ± 1.8b3 20.1 ± 0.5a 16.2 ± 1.1b 64.3 ± 1.2a 38.8 ± 2.4b4 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).

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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_____________________ ________________________

Gene- Highland Lowland Highland Lowlandration C. rosa C. rosa C. rosa C. rosa___________________________________________________________________P 8.8 ± 1.1a 9.1 ± 0.5a 70.6 ± 2.1a 46.2 ± 1.8b1 8.7 ± 0.6a 8.6 ± 1.1a 72.2 ± 1.4a 50.2 ± 1.5b2 10.3 ± 1.1a 10.6 ± 1.2a 72.6 ± 2.5a 54.6 ± 4.6b3 12.2 ± 1.5a 11.9 ± 1.6a 74.2 ± 3.8a 56.2 ± 2.8b4 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 e- Highland Lowland Highland Lowlandration C.rosa C. rosa C. rosa C. rosa____________________________________________________________________P 78.8 ± 10.4a 80.2 ± 8.6a 50.4 ± 1.2b 36.2 ± 0.8b1 74.2 ± 12.6a 78.4 ± 14.4a 54.4 ± 2.6b 34.5 ± 2.6b2 98.2 ± 21.1a 104.5 ± 18.5a 62.1.0 ± 2.4b 44.2 ± 4.1b3 102.5 ± 27.4a 104.2 ± 17.4a 68.4 ± 1.8a 40.8 ± 1.8b4 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).

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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 methodsInsect 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 cloth in a Petri dish. The Petri dishes were immediately transferred to thermostatically

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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 analysisGeneral 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 Student-Newman-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.

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

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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 degree-days (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: 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).

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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.

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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].

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Table 5. Mean ± SE survivorship (%) of immature stages of highland and coastal populations of Ceratitis rosa at seven constant temperatures______________________________________________________________________________________________________________________________Temperature(°C) Egg Larva Pupa

____________________________ ______________________________ _____________________________Highland Coast Highland Coast 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].

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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.

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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

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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±1.4Highland C. rosa + C. fasciventris 15.0±2.1 2.7±1.2 1.0±1.0 16.0±0.5Lowland 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±0.02Highland C. rosa + C. fasciventris 57.8±1.8 0.75±0.01 0.02±0.01 -0.08±0.01Lowland 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♂).

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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 durationCombination (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 ± 23.5Lowland – Highland 21.46 ± 3.50 365.2 ± 18.2Lowland – Lowland 18.76 ± 2.54 311.9 ± 30.1

Highland C. rosa – C. fasciventris Highland – Highland 11.55 ± 4.22 315.6 ± 41.7Highland – C. fasciventris 21.13 ± 4.71 365.2 ± 18.2C. fasciventris – Highland 17.17 ±5.14 333.8 ± 22.6C. fasciventris – C. fasciventris 26.11 ± 4.76 373.2 ± 25.5

Lowland C. rosa – C. fasciventris Lowland – Lowland 20.42 ± 3.50 365.2 ± 18.2Lowland – C. fasciventris 17.67 ± 2.65 309.4 ± 16.9C. fasciventris – Lowland 12.17 ±4.11 343.1 ± 30.8C. fasciventris – C. fasciventris 16.22 ± 3.76 311.4 ± 17.5

__________________________________________________________________________________________________________

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Bactrocera cucurbitae1. 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 Elson-Harris 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

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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 habitatsColonies 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.

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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 partnersDuring 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 – USA2. Dr. Marc De Meyer - Belgium3. Dr. Lucie Vanickova – Czech Republic

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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 5.2Cucumis melo L (Melon) 113 14.2 7.5 1.5 0.0Cucurbita maxima Duch (Pumpkin) 48 100.5 24.8 4.2 3.8Cucurbita pepo L. (Zucchini) 102 30.7 12.2 1.3 3.7Cucurbita moschata (Butter nut) 134 12.5 10.4 3.1 10.4Citrullus lanatus (T.) Mats (water melon) 51 119.4 58.4 11.3 1.8Lagenaria siceraria(M.) Standl (Calabash) 111 60.2 7.8 1.5 0.0Luffa cylindrica (L.) Roen (Luffa) 142 21.7 6.4 1.3 1.4C. melo var. conomon (Sweet melon) 102 66.2 3.2 0.8 0.0Capsicum frutescens L. (Pepper) 789 1.3 4.4 0.6 2.6Lycopersicum esculentum (Tomato) 131 7.7 56.6 12.1 1.4Solanum melongena (Egg plant) 76 3.5 2.1 1.1 2.8Mangifera indica (Mango) 109 31.6 8.2 0.5 121.4Citrus sinensis (Sweet orange) 93 7.4 2.5 0.5 2.1Citrus reticulata Blanco (Tangerine) 40 2.8 1.5 0.2 1.1Psidium guajava (Guava) 42 3.8 2.2 0.4 14.7

Muranga Momordica charantia L(Bitter gourd) 105 18.9 58.2 10.2 1.8Cucumis sativus L (Ccumber) 131 34.9 11.7 3.1 0.0Cucurbita maxima Duch (Pumpkin) 21 52.8 13.8 4.4 1.2Cucurbita pepo (Zucchini) 171 52.1 32.2 1.8 0.0

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Cucurbita moschata (Butter nut) 111 10.1 9.1 3.6 12.5Citrullus lanatus (T.) Mats (Water melon) 60 125.0 48.5 10.2 1.1Capsicum frutescens L. (Pepper) 601 0.8 10.2 1.1 3.4Lycopersicum esculentum (Tomato) 123 6.4 40.1 6.5 0.0Solanum melongena (Egg plant) 51.9 2.2 4.2 1.8 0.0Mangifera indica (Mango) 78 22.1 4.8 0.0 101.2Citrus sinensis (Sweet orange) 50 5.5 2.1 0.5 3.2Psidium guajava (Guava) 58 4.4 1.7 0.2 11.4

Nthagaiya Momordica charantia L(Bitter gourd) 121 22.3 44.8 9.8 0.0Cucumis sativus L (Cucumber) 77 24.8 12.5 3.2 0.0Cucurbita pepo (Zucchini) 160 44.8 24.2 1.4 1.5Cucurbita moschata (Butter nut) 98 11.8 11.3 2.6 10.5Citrullus lanatus (T.) Mats (water melon) 71 138.2 38.2 8.5 0.0Capsicum frutescens L. (Pepper) 923 1.5 15.3 1.1 4.3Lycopersicum esculentum (Tomato) 101 5.1 34.2 5.7 1.1Mangifera indica (Mango) 82 23.5 3.2 0.4 78.9Citrus sinensis (Sweet orange) 66 6.1 4.2 0.0 1.1Citrus reticulata Blanco (Tangerine) 53 3.1 0.0 0.0 2.1Psidium guajava (Guava) 62 4.1 2.2 0.4 10.6

_________________________________________________________________________________________________________________

Bc = Bactrocera cucurbitae; Bi = Bactrocera invadens

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Fig. 3. Host preference and fruit infestation by two populations of B. cucurbitae in field cages. A = bittergourd population; B = tomato population

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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 studies4. Others activities as may be suggested and agreed upon during the Tucuman

meeting.