Clausena anisata (Willd.) Hook. f. ex Benth.—Habit

2.2. Host Plants. Clausena anisata (Willd.) Hook. f. ex Benth.—Habit View in CoL : Shrub. Habitat: Forest margins and regenerations, bushland, wooded grassland, 1700–1875 m. Vouchers: SAJIT–004600, Watuma BM W0069 (EA, HIB), Mwachala G et al. EW012, Faden RB et al. NMK Exped. 263 (EA).

Toddalia asiatica (L.) Lam., Tabl. Encycl. 2: 116 1797. (syn. Aralia labordei H. Lév. ; Cranzia aculeata (Sm.) Oken ; Cranzia asiatica (L.) Kuntze; Cranzia nitida Kuntze ; Limonia oligandra Dalzell (Unresolved) ; Paullinia asiatica L.; Rubentia angustifolia Bojer ex Steud. (Unresolved) ; Scopolia aculeata Sm. ; Scopolia angustifolia Spreng. (Unresolved) ; T. aculeata (Sm.) Pers. ; T. angustifolia Lam. ; T. asiatica var. foribunda (Wall.) Kurz ; T. asiatica var. gracilis Gamble ; T. asiatica var. obtusifoli a Gamble; T. foribunda Wall. ; T. nitida Lam. ; T. rubricaulis Roem. & Schult. ; T. tonkinensis Guillaumin ; Zanthoxylum foribundum Wall. (Unresolved) .

Two wild citrus species, Clausena anisata (Willd.) and Toddalia asiatica (L.) Lam. ( Rutaceae ) were chosen as the host plants for the experiment on swallowtail butterfy, P. desmondi teita developmental attributes. From the forest edges, young branches with new leaves from each host plant were gathered and brought to the laboratory. After washing the branches to get rid of dust and any potential predators, they were submerged in water to prevent desiccation. Te branches were placed in plastic buckets (325 mm dia. × 375 mm ·h) labeled with the host plant species for use in colony maintenance.

2.3. Swallowtail Butterfy, Papilio desmondi teita, Culture. A colony of P. desmondi teita from eggs (collected from an oviposition experiment), larval instars, pupae, and adults was maintained inside the laboratory (situated in Ngangao Forest ) in plastic buckets ( Figure 2 View FIGURE 2 (b)) on C. anisata and T. asiatica . Te stock colony was established from the adults collected at the forest edges in November 2020. Te adults were collected using a sweep net while foraging on the fowers from various plant species, and some females were collected on the selected host plants while laying eggs. To avoid rubbing of the scales on the upper surface of the wings, the butterfies’ wings were folded above their backs before being placed in waxed paper envelopes. After being brought to the laboratory, they were placed in rearing fight cages that were 70 cm by 70 cm by 70 cm in size. A mosquito net was placed over the cages to provide ventilation. Tey

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were also kept from escaping by the nets. Twice a day, they were given 10% sugar solution soaked in cotton wool as food. To ensure successful mating, the males and females were artifcially mated after 24 hours using the hand pairing method [ 18, 30]. Mated females were individually placed into laying buckets containing either young branches of the host plant, C. anisata or T. asiatica , for oviposition.

From the oviposition experiment, all eggs laid on the same day and at the same age on each host plant were collected and placed into clearly labeled transparent plastic

(a) (b)

buckets that measured 325 mm in diameter by 375 mm in height. Tese buckets were then used for incubation and further development in the laboratory. Troughout both seasons, the P. desmondi teita rearing cultures were kept at room temperature and relative humidity. Te mesh-covered, detachable lids on the buckets improved air circulation. After hatching, the young larvae were placed in clean rearing plastic buckets (325 mm Dia × 375 mm ·H) with young, fresh host plants inside, and they were separated from the unhatched eggs using a soft camel-hair brush. Te young, fresh leaves of the selected plants were fed to the larvae.

Te pupae were removed after they had pupated by gently pulling them of and scratching the corner of the silk girdle that the cremaster was attached to. Te pupae were moved to the new buckets, and twigs and white serviette paper were added to the base to support the emerging adults. Up until their adult emergence, the pupae were kept in the same laboratory conditions. After a day, the newly emerged adults were fed with a 10% sugar solution and separated from the pupae. By using the hand pairing method, six pairs were chosen from the population that was reared from each host plant, and they were mated after two days [ 30]. Within the rearing bucket, the mated females were exposed to their host plants in preparation for oviposition. Te second season experiment followed the same procedure used during the frst season.

2.4. Efects of Host Plants on Egg Incubation Period and Mortality Rate. After being separated from the other adults, six mated females were put in buckets with young, fresh branches from each host plant in preparation for oviposition. Eggs of the same age (laid on the same day) were kept in the buckets as mentioned in the previous section, with their lids turned upside down for ventilation, to determine the egg incubation period on each host plant. Until they hatched, the eggs were watched every day. Te incubation period was determined by counting the days until the eggs hatched. To determine the egg mortality rate, the number of unhatched eggs from each host plant was counted. Mortality was calculated as a percentage.

number of unhatched eggs Percentage egg mortality � ×100. (1) total number of laid egg

2.5. Efect of Host Plants on Larval Development Period, Mortality Rate, and Larval Weight. In plastic rearing buckets with adequate ventilation and a branch of young, fresh leaves from the host plants, a cohort of 165 larvae (in the frst season) and 192 larvae (in the second season) was placed. Te duration of larval instar was determined by recording the number of days taken to complete each instar stage identifed by diferences in size, marking and pattern, and head capsule coloration. Te larval weight was determined by weighing 60 individual larvae using JA-SERIES ( JA203 ) electronic analytical balance (0.001 mg accuracy). By monitoring larval cohorts at each instar and keeping track of the number of survivors from the start of each instar to the fnish, the mortality rate was determined. Te mortality rate (by instar) was calculated with the formula

number of dead larvae at the end of instar larval mortality rate � ×100. (2) total number of larvae at the beginning of instar

2.6. Efect of Host Plant on Pupal Period, Pupal Weight, and Mortality Rate. Pupae were marked with the date of formation, and those of the same age were kept together in the rearing bucket and observed daily. Sixty pupae reared on each host plant were used to determine pupal development parameters such as pupal period and pupal weight. During the experiment, the pupal mortality rate was also recorded. Te time taken from pupal formation to the adult butterfy emergence was recorded in days as the pupal period. An electronic analytical balance (Type JA203 H; JA-SERIES) with a 0.001 mg accuracy was used to weigh each pupa. To calculate the pupal mortality rate, the number of dead pupae (those that did not fully transform into adults) were counted and recorded. Abbott’s [ 31] formula was used to calculate the mortality rate :

number of emerged adults pupal mortality rate � ×100. total number of pupae formed (3)

2.7. Efect of Host Plant on Adult Longevity, Sex Ratio, Weight, and Oviposition. By examining the morphology of the forewing, hindwing, and abdomen, adult butterfies were sexed. Te sex ratio was calculated by counting and recording the number of adult males and females. Te adult longevity of males and females fed on 10% sugar solution was investigated by recording the time from each individual emergence to death on a daily basis. Te number of eggs laid in the frst two days was used to calculate the oviposition. After a day, at 6 pm, when they were less active, sixty newly emerged adults were weighed individually on an electronic analytical balance (0.001 mg accuracy; Type JA203 H; JA-SERIES). All experiments were replicated three times and in two seasons .

2.8. Statistical Analysis. All the data were analyzed in R statistical software package, version 4.1.1 [ 32]. Mean and SEM (standard error of mean) were computed for the duration of developmental stages, egg incubation period, oviposition, egg mortality rate, larval and pupal mortality, and weight of developmental stages. Te independent sample t -test was used to compare the means from each host plant. Te Shapiro–Wilk test and the Bartlett test of homogeneity of variance were applied to the fecundity data on various substrates and submitted to one-way analysis of variance (ANOVA) using general linear model (GLM) procedure of R statistical package software. Tukey’s multiple comparison test was used to separate the means between the substrates. P ≤ 0.05 was designated as the signifcance level.