Fig 1.
Spotting on hive fronts increased over time.
The hive fronts of a total of 339 colonies in 3 apiaries were washed with water to remove spots and the presence or absence of spots on hive fronts was evaluated 5 and 10 days later.
Fig 2.
Apis cerana defended their colonies from group attack by Vespa soror by applying fecal spots around hive entrances.
(A) A hive front with heavy fecal spotting around the entrance opening. (B) A marked A. cerana forager on a dung pile. (C) A forager holding a clump of fecal solids in her mandibles, captured after leaving a dung pile. (D) A forager applying a fecal spot to a hive front after being paint marked on a dung pile. (E) An entrance-focused group attack on a colony by six V. soror workers. (F) Damage to a hive entrance after entrance margins were chewed on by V. soror workers (the attack was stopped by experimenters before the nest was breached).
Fig 3.
Fecal spotting on hive fronts increased in response to attacks by Vespa soror.
In two tests, visits by hornets to attacked colonies occurred naturally over the course of a single day; hornets were prevented from approaching control colonies at all times. Hornet symbols signify treatment and time combinations during which hornets were permitted to attack. (A) Test 1: hive fronts were photographed at two-hour intervals during the day of attack and at the start of the next day to assess changes in spot number. (B) Test 2: hive fronts were photographed at the end of a day of attack and at the end of two subsequent days during which hornet attacks were prevented. Spot counts on hive fronts were estimated from photographs. All colonies were in the same apiary, but different colonies were used for each test. Different letters indicated significant differences in mean (± SEM) spot number across conditions.
Table 1.
Vespa soror attackers were more likely to land at and chew on entrances of hives than Vespa velutina attackers.
Visits to Apis cerana colonies by V. soror (n = 857 visits) and V. velutina (n = 328 visits) hornets were observed in the same apiary and over six days per species. Attacks were monitored by observers stationed throughout the apiary and data were recorded only if hornet visits lasted at least 30 seconds and were observed from initial approach until departure from a hive. Asterisks indicate behaviors that are required to breach hive entrances and occupy nests.
Fig 4.
Fecal spotting on hive fronts increased after colonies were exposed to VG extracts.
Each extract mixture was created by placing VGs from three Vespa soror workers into a vial with 0.5 mL ether for 24 hours (one vial per test colony). A 1 cm2 piece of filter paper was repeatedly soaked and dried in this mixture before it was pinned at a colony entrance; ether shams (filter papers without extracts) were created similarly and presented to colonies as a control. Hive fronts were photographed after six hours to determine changes in spot number. Asterisk indicates a significant difference between treatment means (± SEM) by the end of the test (p = 0.02).
Fig 5.
Exposure to VG extracts from Vespa soror workers induced spotting of hive fronts by most colonies.
The addition of new spots to filter papers and hive fronts was determined six hours after colonies were exposed to filter paper soaked either in VG extract or in ether only (a sham control); refer to Fig 4 for details about the creation and presentation of VG extract-soaked and control filter papers. Asterisks indicate a significant difference between comparison groups, which are identified by brackets (* p ≤ 0.03; ** p = 0.001).
Fig 6.
Vespa soror workers spent less time trying to breach nest entrances as spotting increased.
Images show the spread of spots around entrances for colonies that ranked at the midpoint of each of the three spotting categories: (A) light spotting (median 42, range 8–99 spots per hive front); (B) moderate spotting (median 275, range 107–495 spots per hive front); (C) heavy spotting (median 636, range 504–1,523 spots per hive front). White scale bars = 5 cm. (D) Mean time (± SEM) per hornet spent visiting a colony and, for hornets that landed on hives, time spent performing nest-breach behaviors (i.e., landed anywhere on the hive, landed at the entrance specifically, chewed on the entrance margins). Visit duration was calculated for all hornets (left: light = 81 visits; moderate = 141 visits; heavy = 54 visits). Time spent trying to breach nests was calculated only for hornets that landed on hives (right: light = 72 visits; moderate = 115 visits; heavy = 46 visits). Data were obtained from the videos used to produce Table 2 (see legend). Asterisks indicate a significant difference among groups identified within brackets (** p < 0.0001); letters indicate differences among treatment means).
Table 2.
Moderate or heaving spotting reduced multiple-hornet attacks and nest breach behaviors by Vespa soror workers.
Data were obtained from videos of hive visits by V. soror workers that were recorded in the same apiary over a three-day period (the source of data for Fig 6). For landing, chewing, or killing, data were collected from videos for individual hornets to determine the percentage of all hornets that performed these attack behaviors while visiting colonies that were lightly (n = 81 hornets), moderately (n = 141 hornets), or heavily spotted (n = 54 hornets). For incidence of multiple-hornet attacks, the percentage of videos that recorded attacks on lightly (n = 61 videos), moderately (134 = videos), or heavily spotted (n = 47 videos) colonies that included more than one hornet was determined (here, each video was a replicate). Because a single video could yield data for several hornets if it recorded a multiple-hornet attack, the total number of attack videos was lower than the total number of hornet visits. Asterisks indicate behaviors that are required to breach hive entrances and occupy nests.