Adaptive Defence Strategies of the Stingless Bee, Tetragonula Iridipennis Smith. Against Nest Intruders in a Newly Divided Colony

The study was conducted at the experimental farm of the ICAR-National Bureau of Agricultural Insect Resources (NBAIR) Bengaluru, Yelahanka Campus (13.096932N, 77.56759E) which is located in the heart of the rapidly growing capital city of the South Indian state of Karnataka.

Stingless-bee colonies were maintained in wooden square box hives (17.5 cm × 17.5 cm × 11.5 cm) in the meliponiary located at the study site. Colonies with more than fifty pollen and honey pots with a higher volume of larval and pupal brood cells with relatively higher hive traffic of incoming and outgoing foragers were considered strong. The identified strong colonies were formed through the collection of approximately 25–30 pupal brood cells, a queen cell, ten pollen pots and honey pots and two scoops of resin from the strong colonies and placed in a new wooden square box hive. Fifty foragers were also added to each rectangular hive during the division. Ten replicates of similar colonies were maintained for our observations. A small hole 1.0 cm in diameter was made in the bottom of the newly used wooden square box hive, and a polyethylene transparent tube (1.0 cm in dia.) was inserted to serve as an entry/exit for foragers. Resin was also applied to the outer surface of the polyethylene transparent tube to enable outgoing foragers to navigate back to their nest in the newly divided colony.

Ten newly divided colonies of stingless bees were selected and established colonies were monitored weekly for attack by invaders. The observations were recorded for a period of twenty-eight days after division from 7.00 am to 6.00 pm, as this is a crucial period for the initial acceptance of the new hive and colony establishment. The invading arthropods were collected, pinned, preserved and taxonomically identified. The nature of the invaders and the number of attacks per week for a period of four weeks were recorded. The number of nest closure events per month, mode of entrance closure and material used for the closure of the entrance were also recorded. The period of nest closure and time taken for guard bee activity was recorded for every 24-hour interval for a period of one week. The initiation of foraging activity was recorded three days after the division of the colonies. The change in the architecture of the inner hive after the invasion of the enemy was also recorded.

The bees introduced during colony division were monitored at an interval of two hours from the date of division for a period of two weeks by opening the top lid of the hive during the nest closure period. The activities of the introduced bees within the hive were recorded. The number of days needed for nest closure, guard bee activity at the hive entrance and forager activity were also recorded. The percentage of bees undertaking the initial colony establishment activities of in-hive architecture formation, hive entrance repair and tending the emerging bees from the brood cells were recorded.

An experiment documented the nature and extent of damage caused by intruders in the newly divided colonies. Two treatments were used for the study, one set of newly divided colonies exhibiting natural defence against the intruders and a control set with of a newly divided colony where natural defence mechanisms exhibited by the colonies were excluded. The closure of the nest entrance, the formation of a complex nest entrance and the resin applied to the entrance tube were considered as the natural defence mechanisms the stingless bees. The natural nest defence mechanisms exhibited by the bees were removed through the dismantling of the nest entrance tube with the use of a fine needle to allow the invaders into the colony. The effect of the intruder's attack on brood emergence and the robbing of storage pots in both treatments were recorded for a period of twenty-one days.

Data on various parameters studied were statistically analysed. The mean ± SD of the number of bees repairing the hive, guarding and tending the brood was calculated. Analysis of variance (GLM in SAS 9.3; SAS Institute, Cary, NC) was used to compare the number of invasions in the first four weeks after the division of the colonies with the effect of colony defence over the percentage of brood emergence and the percentage of resource robbery. Where a significant difference was detected, treatment means were separated using the Tukey HSD Test (0.5%).

The newly divided colonies of stingless bees were observed to be invaded by two species of solitary resin bees (Fig. 1), Megachile cephalotes, M. disjuncta, two species of ants Camponotus sp. and Oecophylla smaragdina, and an unidentified spider species belonging to family Salticidae (one female and one spiderling), wolf spider, Pardosa sp. (Lycosidae: Araneae). A parasitic wasp species Leucospis sp. was observed near the nest entrance (Fig. 2). We did not observe nest entry/invading activity by the parasitic wasp.

Fig. 1.

Fig. 2.

The sequence of activities during the initial stages of colony division are presented in Fig. 3A–D. The foragers sealed the nest entrance with a thin layer of yellowish-brown coloured resin which restricted entry by invaders. Complete nest closure was sometimes observed at the outer part of the entrance tube and sometimes at the inner part of the entrance tube which protruded into the colony. The behavioural response of the stingless bees in the colony to invasion from different natural enemies was is in Tab. 1. Resin bees stole resin at the nest entrance of the newly divided colonies. In two colonies, resin bees were trapped in the polyethylene tube fitted in the entrance and died from a lack of further movement and asphyxiation. We also observed a deposition of fresh brown-coloured sticky resin droplets at the entrance in the mud pot hives during the hiving of wild stingless-bees colonies, which is a direct defence strategy to trap any intruder in the colony.

Fig. 3.

A significant difference was observed in the invasion by resin bees (F_3,15=5.90; P<0.0001) during the four weeks after colony division (Fig. 4). The highest mean number of resin bee invasions was recorded during the first week of colony division (11.67±1.53 invasions) followed by a relatively lower number of attacks during the second (2.00±0.31 invasions), third (1.15±0.67 invasions) and no invasion four weeks after division. No significant difference in spider invasion was observed during the four weeks after colony division. The mean number of spider attacks was highest during the second week after division (4.67±1.04 invasions) followed by 3.00±1.00 invasions during the third week and 1.00±0.57 invasions during the fourth week after division. Ant invasion in the colony differed significantly during the four weeks of colony division (F_3,15=8.19; P<0.0001). The mean number of ant invasions during the first week (4.33±1.15 invasions) after colony division was the highest followed by the second (1.02±0.16 invasions), the third week (1.00±0.08 invasions) and no invasion four weeks after colony division.

Fig. 4.

Nest closure was observed for a period of 4.37±0.74 days after the division and the guard bee activity and outgoing activity of foragers for resource collection for periods of 6.13±1.24 days and 10.63±1.07 days, respectively (Tab. 2). Of the fifty introduced bees in the newly divided rectangular hive, 18.40% participated in the initial activities of hive architecture, namely hive entrance repair, attachment of the brood cells using resinous pillars and forming involucrum to protect the brood cells. Around 69.20% of them were observed to participate in the hive entrance repairs by completely rebuilding the entrance with a resinous papery multilayered tubular material. The bees were also observed to tend to the new emerging bees from the given pupal brood cells (13%) in the colony (Fig. 5).

Fig. 5.

Among the three major invaders of the newly divided colonies, ants and resin bees were observed to rob resources and damage the brood cells (Fig. 6). In divided hives where natural defence strategies were excluded, ants were seen consuming the brood in large numbers, negatively impacting the brood emergence rate. The rate of emergence for the brood varied significantly between the newly divided colonies with intact natural defence and the colonies with excluded defence (F_1,14=55.20; P<0.0001). The percentage of emergence for the brood in the divided colony that exhibits its defence mechanism and the colonies where the natural defence mechanism was excluded were 100 and 10.1%, respectively. Ants were also observed to rob the honey pots leaving the pollen pots intact. A significant difference in the percentage of ants’ robbery of honey pots was recorded between newly divided colonies with intact natural defence mechanisms and colonies with excluded natural defence (F_1,14=108.12; P<0.0001). We did not observe any robbing of honey pots in the colonies with intact natural defence mechanisms whereas, in natural defence-excluded colonies, a significantly high rate of resin robbing (94%) was recorded. A newly divided colony invaded by ants could be clearly seen by the movement of ants at the entry and exit points of the colony. In the colonies excluded from the defence mechanism, the resin bees were found to have completely robbed the resin material but leaving the brood cells and storage pots intact. No initial hive-building activity was observed in such invaded colonies. Spiders were seen to form a web inside fully damaged colonies two weeks after division in the colony devoid of resources, namely honey and live brood cells.

Fig. 6.