Feeding Frequency and its Related Effect on Productivity and Survival Rate of Glossina Pallidipes in Kality Tsetse Mass Rearing and Production Center for the Purpose of Sterile Insect Technique (SIT)

S. Feeding Frequency and its Related Ef fect on Productivity and Survival Rate of Glossina Pallidipes in Kality Tsetse Mass Rearing and Production Center for the Pur pose of Sterile Insect Technique (SIT). Bi omed

Island, Zanzibar [6,7]. As a consequence of this success, programs were developed to apply this approach on the African mainland and, in 1996, the government of Ethiopia embarked on such a program to create a zone free of Glossina pallidipes in the Southern flies and will be able to produce over 700 000 sterile male flies per week enough to treat approximately 7000 km 2 at a time [8].
However, the major constraint in these early programs has been the large scale production of sufficient sterile male flies for release.
The establishment and expansion of G. pallidipes colonies for mass production of sterile male flies proved to be difficult and they collapsed several times due to the deleterious effect of lack of quality blood and good feeding management, high infection rates with the salivary gland hypertrophy virus, unsuitable environmental factors even if it requires the mass production of males for sustainable sequential release until major population reductions are achieved [9][10][11][12][13].
Tsetse rearing evolved from feeding on live hosts to an in vitro rearing system where blood is fed to flies through a silicone membrane [13]. Both males and females feed on blood. The female reproduces by adenotrophic viviparity, one larva at a time being nourished in utero by a secretion from the uterine gland [14]. This is very important since both males and females (also including the larvae within the uterus) depend on the blood for their survival and nourishment. The amount of blood imbibed during the interlarval period is the major factor determining the productivity and survival rate of tsetse, Glossina species. Therefore, qualitative and quantitative amount of blood is needed for the maintenance of fly colonies [15] in vitro. In vitro feeding system is recommended if a reliable source of quality tested blood is available [16][17][18]. However, in a mass-rearing facility, the logistics of obtaining sterile, highquality blood remains problematic. So, appropriate and economical use of blood in the membrane (in vitro) feeding technique provides a means to produce tsetse flies more economically and with less risk but this has not been established for G. Pallidipes in Ethiopia.
Therefore, the objective of this study was to establish the optimum feeding frequency and regime required for tsetse mass rearing and production colonies of G.pallidipes.

Study Area
The present experimental study was conducted at Kality

Study Period and Population
The study was conducted from March 25 to June 20, 2019, and

Study Design
Feeding Frequency Test: Two experimental groups were established and reared based on feeding frequency regimes as the feeding of three and five times a week. Each group has six replicates of a cage with 48 female and 12 male flies per cage called colonies using zero-day mating immediately after they had emerged from incubated pupae using a cooler (chiller) set at +40C and were maintained in a replicated cages diameter of 20 cm and width of 5 cm netting on top and bottom for feeding and collection of the hatched larvae [19]. All twelve established colonies were again allocated into two groups (six colonies in each group) to be subjected to different feeding regimes during the subsequent pregnancy cycle for three consecutive months. One group was fed three times per week on 1 st , 3 rd and 5 th and the second group was fed five times on 1 st , 2 nd , 4 th , 5 th and 7 th days per week.
These two feeding regimes were selected based on the former one has been used as a feeding option in the facility, while the second one revealed what reproductive performance a tsetse fly can achieve on the highest possible feeding regime. Pupae were collected daily and mortality checks were done every week. The weekly datasets of the G.pallidpes colonies, in different feeding regimes, were measured by observation of tsetse production parameters including fecundity, Pupae Per Initial Female (PPIF), mortality rate and pupae production and pupal size class graded based on their size. Both experimental groups of Flies were fed from a similar quality of blood factor from the same batch of defibrinated gammairradiated bovine blood meal using an in vitro silicon membrane system which was previously collected aseptically and frozen at -20 °C [12,20]. The feeding system was installed in a climatic room that was maintained a similar environmental condition of 25 ± 1 °C and 50 ± 5 % humidity for all treatments. The flies remained in the feeding room for less than 30 minutes based on [21,22] and placed under similar and optimum environmental conditions. The holding room temperature and humidity was adjusted (controlled) and monitored daily from the data logger set in the rearing room. The only difference between treatments was the feeding frequency of three times a week (Monday, Wednesday and Friday) and five times a week (Monday, Tuesday, Thursday, Friday and Sunday).

Productivity Testss
Fecundity Test: Mating cages were placed in individual larvipositioned cups and Pupae were collected from all cages daily in the morning beginning from days 16 up to the end of the experiment. All data were recorded separately per tested groups per cage and analyzed for three continuative months including their pupal size which was graded as indicated in Annex 5 [16]. adjusted to correspond the five weight classes previously has been defined to tsetse pupae of G.pallidipes [16].

Mortality Test: Mortality was recorded daily (except on
Saturdays and Sundays) for each test group until the death of the last individual. Dead flies were sorted into blood-fed and starved fly mortalities [16].
Data Analysis: To optimize the weekly feeding operation (duration of feeding and frequency of feeding) for G. pallidipes, two different combinations of feeding frequency were tested using defibrinated gamma-irradiated bovine blood. For both groups, the two feeding tests were continued for 17 weeks. All statistical analysis was performed using STATA software version 12 (Stata Corp, Texas, USA) [23]. Pair-wise comparisons of median survival between test groups were estimated using a Tukey'spost-hoc test ('glht' function in the 'multcomp'package). Feeding frequency was used as explanatory variables and survival rate as the response variable. The pupal production and the mean of pupal size were analyzed using general linear models [21]. The smallest AICc of different models was used to choose the best model and the significance of fixed effect was tested using the likelihood ratio test.
Pair-wise comparisons of median fecundity between treatments were tested with a Tukey'spost-hoc test ('glht'function in the 'multcomp'package). Female productivity was estimated as pupae per initial female (PPIF, the total number of pupae produced in a given time divided by the number of initial females). The unit 'PPIF' is commonly used to assess the health of the Glossina colonies.
Female productivity was measured as pupae per initial female (PPIF, the total number of pupae produced in a given time divided by the number of initial females).

Pupae Per Initial Female and Fecundity
To optimize the weekly feeding operation for G. pallidipes tsetse flies and maximize their production in the facility, two different combinations of feeding frequencies were tested (three times a week and five times a week). For both test groups, the two feeding treatments were continued for 17 weeks. A 10-week comparison of flies maintained and reared fewer than two feeding frequency with the standard round cage with 48 females and 12 males showed that productivity was varied greatly between the two test groups. The overall commutative Pupae Per Initial Female (PPIF) and standard deviation of flies subjected to different feeding regime of five and three times per week were found to be 3.05±0.47, and 2.21±0.4 respectively both starting from their first lariviposition date which was 16 days later respectively (Figure 1 and table 1). (Figure 1) G.

Pupae Size Determination
The pupal size categories for G. pallidipes pupae produced under two feeding frequency five vs. three times per week are given in Figure 6. The results were estimated as a percentage of the total pupae in size categories A-E. Pupae classes represented by letters A to E indicated size in ascending order where A refers to small pupae while E refers to large pupae. (Figure 6) Overall

Percentage of G. pallidipes pupae in different size categories (A-E)
between test groups. (Table 2) Levels of significant differences among proportions of pupae size of G. pallidipes flies between tested feeding frequencies analyzed using multi linear regression.
( Figure 7) Overall Percentage of G. pallidipes pupae in various size (Grade) categories (A-E) between tested feeding frequencies.

Mortality Rate Accumulated and Daily Mortality Rate of G.pallidipes
The highest overall commutative mortality was recorded in flies fed three times per week (54%) compared to flies fed five times per week (47%) over 10 weeks (Figure 4). The lowest daily mean mortality rate was also recorded on flies fed five times per week (0.6%) compered to three times per week (1.4%) ( Figure 5). The mean daily survival rate of the female G.pallidipes was significantly varied with the frequency of feeding regime of blood supplied per week (t=-2.03 P=0.045) 3.2. Effect of feeding frequency on pupae quality. (Figure 4) Accumulated percentage of dead females G.pallidipes subjected to both feeding regimes. (Figure 5) Percentage of Mean daily mortality of females G.pallidipes subjected to both feeding regimes.    week. Even without ATP, blood that had been frozen enabled tsetse flies to achieve a high level of productivity. However, Glossina morsitans submorsitans Newstead easily tolerated a reduction from six to three days per week (Monday, Wednesday, and Friday) without reducing female productivity. According to the findings of [24,25,26], the amount of blood imbibed during the interlarval period is the major factor determining the reproductive performance of tsetse, Glossina spp., whether measured as the size or number of pupae produced. There was equally and well-established fact that in the laboratory it is necessary to allow tsetse colonies the opportunity to feed at least every other day to ensure the regular production of pupae [25,7] although when offered a meal every 1 or 2 days not all flies choose to feed at every opportunity, giving a mean feeding interval in the laboratory of between 2 and 2.5 days [27,28]. However, this result is different from previous experiments showing that G. brevipalpis colony flies be fed for 5 min three times per week was the most practical and economical solution concerning available labor and volume of blood used [29]. These facts reflect the importance of storing up nutrients ingested early in pregnancy for later use in larval development, for which meals on days 1, 4 and 7 are apparently insufficient in the laboratory. A meal taken only on day 1 is sufficient for normal development up to day 5, but cannot meet the demands of rapid larval growth from day 6 onwards. In the laboratory female flies which take a full blood meal every third day produce fewer, smaller pupae than those which take on average rather smaller blood meals every second day, and show at what stage in the pregnancy cycle larval development is adversely affected by a nutritional shortfall. Although a reduction in feeding frequency from four to three times per pregnancy cycle resulted in a few abortions apparent from day 6 onwards [22,[30][31][32]. However, the PPIF required for the establishment of a colony should be ≥3, and thus the values obtained for feeding frequency of three times per week colonies were usually rather low [29].
Similarly, the highest overall commutative mortality was also recorded in flies fed three times per week (54%) compared to flies fed five times per week (47%) over 10 weeks. The lowest daily mean mortality rate was also recorded on flies fed five times per week (0.6%) compared to three times per week (1.4%). The mean daily survival rate of the female G.pallidipes was significantly varied with the frequency of feeding regime of blood supplied per week (t=-2.03 P=0.045). To have a steady growing tsetse colony, it was important to make sure that the colony's daily mortality was kept below 1% and the fecundity p/f10 was above 0.5 [29]. A major difference between field and laboratory-held flies is in the size of the blood meal taken; in our laboratory, flies held individually and fed on rabbits every third day take about 78% of the blood meal recorded for field flies. It is not surprising, then, that three feeds were sufficient only for a production rate of 0.70 pupae per cycle.
The flies in Langley & Stafford's [25] study were membrane-fed on defribinated pig blood, which results in an even lower meal size, lower survival rate, lower fecundity and smaller pupae than when flies are fed on rabbits [20]. Based on the laboratory results, therefore, we can only conclude that tsetse requires at least four or five laboratory-type meals per pregnancy cycle to reproduce satisfactorily, which cannot necessarily be taken to mean the same number of meals in the field.
Pupal quality as measured by weight and converted to size classes showed that the distribution of the pupae percentage in the different size classes produced by females G.pallidipes maintained under both feeding regime diets was found to be different in both feeding frequency. Pupae produced in under feeding frequency of three times per week fell mostly into the smaller size categories A-C compared with pupae maintained an under feeding frequency of five times per week which fell mostly into categories C-E but statistically significant variation were not recorded in all class except D class pupae. Furthermore, pupae quality decreases as the flies getting older and older. The highest percentage of pupal quality class (class D)( 48% was recorded in flies fed five times per week (48% )compare to other feeding regimes (35.9%).This implies that flies fed five times per week gave the high quality of pupae than flies fed three times per week. However, five times feeding regime had a superior effect on the production of quality pupae than other feeding regime [33] found that the weight of a pupa depends on the amount of blood taken by a female during pregnancy, with a highly significant correlation between puparium weight and quality of blood feed. These results were confirmed by Jordan et al. who found that heavier pupae were produced by well-nourished females [34].

Conclusion
Finally the study concluded that feeding frequency greatly affected both the productivity and survival rate of G.pallidpes reared under laboratory conditions. The survival and pupae production of G. pallidipes flies appeared to be governed mainly by their feeding frequency, and require at least four or five laboratory-type meals per pregnancy cycle to reproduce satisfactorily and five laboratorysized meals may be sufficient to meet the energy demands of normal larval development in the laboratory which increased their productivity compared to three-sized meal.