Insect visits and variation of floral temperatures during blossoming of Rafflesia gradutensis

. The survival of parasitic plant Rafflesia depends on the host plant, habitat condition, vegetational types, , pollination and the dispersal of seeds. This study aims to investigate visits by insects and variation in floral temperatures of Rafflesia gradutensis during flowering. The flower was observed to be visited by green fly Lucilia sericata and weaver ant Oecophylla sp. The number of visits L. sericata was 617 times and 337 times by Oecophylla sp, in which most of the visits occur during mid-day. The average observed highest surface temperatures of were 28.4 0 C (perigone) and 28.7 0 C (diaphragm), which were slightly above the air temperature of 27.8 0 C. The temperature experiences a gradual increase from the morning till the noon, and then it decreased toward late afternoon. The results of the floral temperatures indicate endothermy in R. gradutensis . However, thermo-regulation related to endothermy needs further investigation.


Introduction
There are three genera included in a family Rafflesiaceae, all of which are holo-parasites with a specialized host, i.e., vines in the genus Tetrastigma.Members of Rafflesiaceae have a unique growth habit.The flower is the only plant organ that is seen outside the host.They do not have leaves, stems, and roots.About 36 species of Rafflesia have been identified, occurring in Indonesia, the Philippines, and Malaysia [1].The genus Rafflesia is known to be the largest flower of the plant kingdom [2].Rafflesia arnoldii produces the world's largest flower, which is up to 1 meter wide.
Rafflesiaceae is distributed in South East Asia from Thailand through western Malaysia and the Philippines, Borneo, Sumatra and Java [2,3,4].As this region suffers huge loss of forest area, some habitats of Rafflesiaceae have also been destructed.Sumatra, home of 11 species of Rafflesia, has been rapid and potentially represents a serious risk to native species [5].Consequently, a member of Rafflesiaceae has been being threatened with extinction [5,6].Therefore, the sustainable conservation of Rafflesiaceae is urgently needed.
The life of Rafflesia is determined by several factors.Parasitic Rafflesia fully depends on the Tetrastigma species as host plant.Other factors which determine the proliferation of this species are vegetation types and micro-climate, edaphic factor, topography, pollination and seed dispersal as well as human activities [1,7] .As Rafflesia is dioecious plant, the role of pollinators is crucial [8].However, the knowledge of reproduction biology of Rafflesia, including pollination, needs more investigation.This knowledge is important in conserving Rafflesia both in-situ and ex-situ [9].
Insect species is believed to be possible agents for pollination in Rafflesia [8,9].Insects may be attracted to come to Rafflesia due to smell and visual appearance of the flower.The release of odour by flowers is related to endothermic mechanism, which may due to internal heat by the flower.However, little is known the mechanisms of flowers to heat and how the heating is regulated in response to changes in ambient temperature [10].One species member of Rafflesiaceae, Rhizanthes lowii is a strongly endothermic flower that exhibits weak thermo-regulation [11].As endothermic is related to the odour release by the plant, it may play an important role in attracting pollinators to come; hence facilitating fertilization to occur.Therefore, more information on insect visits and also the flower temperature of Rafflesia could deepen our understanding of pollination biology of this enigmatic flower.This study aims to investigate visits by insects and variation in floral temperatures of Rafflesia gradutensis during flowering.
The object of this research is Rafflesia gradutensis, which is one of the five Rafflesia species occurs in Bengkulu.The variation of the flower temperature was done by measuring surface temperature using infra-red thermometer of perigone lobe and diaphragm of the flower in five different time, i.e., 06:00 am, 09:00 am 12:00 pm, 15:00 pm and 18:00 pm.The temperature was measured every 5 minute for 30 minutes for each designated observation time.Insect visits were observed by direct observation and taking picture of the flower for 30 minutes for each designated observation time.The visiting insects were identified and the number of visits were recorded.The environmental factors, i.e., air temperature, humidity and light intensity were also recorded.All the observations were taken every day during blossoming period of R. gradutensis for 6 days.

Results and discussion
The average of daily air temperature and humidity are very much consistent with the pattern of the day.The highest temperature was about 27.85 0 C and the lowest humidity was about 79.46 during the mid-day (Table 1).The maximum light intensity reaching the forest floor was about 55.87 (W/m 2 ) at 12:00 and the lowest light intensities was early morning and evening (Table 1).The area is also very much shaded by tree canopy.It took 6 days for the observed R. gradutensis to fully blossoming and reached its full size of the flower (53 cm in diameter).Two species of insects were spotted to visit R. gadutensis during the blossoming period, i.e., green fly Lucilia sericata and weaver ant Oecophylla sp (Figure 1).Insects may play an important role in pollination biology of Rafflesia [1].Insect species of Lucilia and Chrysomia were found as possible pollinators for R. pricei [8].Insects were found to visit R. kerrii and R. patma include Chrysomya, Lucilia, and Sarcophaga [13,14].In total, the number of visit by Lucilia sericata was 617 times and 337 times by Oecophylla sp during the 6-day of blossoming (Figure 2).The most visits occur during midday, which were in average 77.2 visits by Lucilia sericata and 21.8 visits by Oecophylla sp every day.The least visits recorded during early morning and late afternoon.The pattern of the visits by two species of insects are very much related to the pattern of floral temperature of R. gradutensis.The floral temperature varied during the course of the day, with the highest temperatures occurred during the mid-day.The average temperature at 12:00 pm was 28.4 o C for perigone and 28.69 o C for the diaphragm (Figure 3), which were slightly above the air temperature (27.8 0 C).The average temperatures of perigone and diaphragm were not significantly different.It cannot be determined if the variation of floral temperature was due to internal thermoregulation or environmental regulation.The pattern of floral temperature was similar to air temperature; hence, the increase of floral temperature may be more related to the change in air temperature and the decrease of humidity.However, as the floral temperature increase, the odour of the flower may be more distinct.Based on other studies, endothermy was detected in another species of Rafflesiaceae, i.e., Rizhantes lowii.The sign of endothermic regulation in R. lowii was detected by fairly constant temperature during and after flowering [11].Another species, i.e., Rafflesia tuan-mudae also showed sign of endothermy, although it is only a weak pattern and there was no sign of thermo-regulation.R. gradutensis may exhibit endothermy as it emits odour; however, the establishment of internal thermoregulation in R. gradutensis needs more investigation.
As insect species may play role as possible agents for pollination in Rafflesia [8], the characteristics of Rafflesia's flower to attract insect to come become crucial.Insects may be attracted to come to Rafflesia due to smell and visual appearance of the flower.Flies were very common insects found in many studies to visit Rafflesia, which may be more attracted to the smell of the flower.Flies recorded to visit both male and female flowers [8,14].The insects may stay long enough to carry or to transfer the pollen from one flower to another.Five genera of flies were spotted visiting a female flower of R. patma [9].This research results suggest the variation of floral temperature is related to the number of visits by insects.As insects may play a role as pollinators, the time in which the visit mostly occurs is probably the best time for pollination.

Conclusion
Two species of insects were observed to visit R. gadutensis during the blossoming period, i.e., green fly Lucilia sericata and weaver ant Oecophylla sp.Lucilia sericata individuals were recorded to visit R. gradutensis for 617 times and 337 visits for Oecophylla sp during observation.Most of the visits happened during mid-day.The floral temperature varied during the course of the day, with the highest temperatures occurred also during the mid-day.The average temperature at 12:00 pm was 28.4 o C for perigone and 28.69 o C for the diaphragm.The variation in floral temperatures may indicate endothermy in R. gradutensis.This endothermic mechanism allows flowers to emit odour; which then attract insects to visit.However, thermo-regulation related to endothermy needs further investigation.As insects may play a role as pollinators, the time in which the visit mostly occurs is probably the best time for pollination.

Fig. 2 .
Fig. 2. The number of visits by green fly Lucia sericata and weaver ant Oecophylla sp during blossoming period of R. gadutensis.

Table 1 .
The average of daily air temperature, humidity and light intensity at the area of observed R. gradutensis.