Emergence timing and voltinism of phantom midges, Chaoborus spp., in the UK

After introduction of overwintered fourth instar larvae (2027 in total), emergence timing of adult Chaoborus spp. (Diptera: Chaoboridae) was investigated in four outdoor freshwater microcosms in the UK in 2017. Adults started emerging on 13 April and emergence reached a peak on 2 May. The majority of emergence was completed by 3 June. Emergence rates for each microcosm ranged from 51.4% to 66.2% with a mean of 60.9%. The great majority of emerged adults were C. obscuripes (99.68%). Males appeared to emerge slightly earlier than females. The results indicated that for overwintered C. obscuripes larvae, the adults emerged en masse in spring (rather than emerging gradually over the course of spring and summer). In a separate experiment at the same location, the number of Chaoborus spp. life-cycles occurring per year was determined using six replicate groups of microcosms, each group containing four microcosms. Each microcosm contained 200 L of water and was enclosed within a ‘pop-up’ frame covered with ‘insect-proof’ mesh (1 mm2 aperture). The first microcosm in each group was ‘seeded’ with egg rafts (first generation) of Chaoborus spp. Following adult emergence, as soon as the first egg rafts were laid in each microcosm these were removed and transferred to the second microcosm in that group, and so on. The larvae sampled from the second and subsequent generations in the microcosms were all C. crystallinus. C. crystallinus produced up to four discrete generations within the experimental period, and life-cycle times from egg-to-egg ranged from 14 days (replicate group 5, first generation) to 56 days (replicate 3, second generation). These two experiments, indicated that i) adult C. obscuripes arising from overwintered larvae emerged en masse in the spring, and ii) up to four generations of C. crystallinus occurred; i.e. C. crystallinus exhibited a multi-voltine life history under the temperate conditions of this UK study.

172 Active microcosms in both trials were monitored weekly for temperature, pH, dissolved oxygen and 173 conductivity using a Hach HQ40d portable multimeter . Water temperature in one unit was monitored 174 with readings every 30 minutes, using a calibrated data logger. Additionally, the water temperature in 175 an unused, unenclosed microcosm was also monitored continually from June onwards, to allow a 176 comparison of temperatures in enclosed and unenclosed systems. Climatic conditions on the 177 microcosm site were recorded throughout the study using a Davis Vantage Pro2 Plus field weather 178 station, situated approximately 100 metres from the study area.

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215 Each of the four microcosms established for the production of egg rafts were initiated with 216 approximately 500 4 th instar Chaoborus spp. larvae from the same source as used to initiate the 217 emergence experiment and covered with insect-proof netting. Following the emergence of adult 218 Chaoborus spp., these egg generation microcosms were regularly monitored for the presence of egg 219 rafts on the water surface. When egg rafts were found they were transferred to the first of the four 220 microcosms in each replicate set. The production of egg rafts in the egg generation microcosms was 221 monitored until no more egg rafts were required. Each of the first of the four microcosms in each 222 replicate set containing egg rafts was inspected at least three times each week for the appearance and 223 development of larvae, pupation, emergence of adults and deposition of egg rafts. The presence of 224 larvae and their approximate instar (estimated by eye) together with the presence or absence of pupae 225 and emerged adults was recorded by inserting a 19 cm diameter white disc attached to a rod to Page 11 of 20 226 provide contrast for assessing both at the surface (early-instar larvae) and at depth (late instar larvae 227 and pupae) and visual inspection of the enclosure mesh (adults). The date and numbers of any egg 228 rafts produced were also recorded.
229 Emerged adult insects were allowed to remain within the enclosure, reproduce and deposit egg rafts 230 on the water surface. These eggs were then collected and added to the second microcosm of each 231 replicate set to initiate populations. Established microcosms were inspected three times per week until 232 the end of September when the monitoring frequency was reduced to once per week. The presence or 233 absence of each life stage of Chaoborus was recorded in each active microcosm. Where present, an 234 approximation of the size range of larvae visible was recorded, mainly to facilitate monitoring of egg 235 hatching success and the rate of development, to ensure that critical development stages were not 236 missed. Once adult emergence had been observed, at each subsequent assessment, the water surface 237 was inspected for the presence of egg rafts deposited by emerged females. The observation of the 238 first deposition of egg rafts was recorded and those egg rafts used to initiate the next sequential unit 239 within the replicate. Subsequently, additional egg rafts produced within the active units were also 240 transferred to supplement the next unit's population, until it was considered that no more were 241 required. 242 On three occasions, once in July and twice in October, samples of late-instar larvae were taken and 243 preserved in 70% alcohol for identification to species level. In the July sampling, only three 244 microcosms contained larvae considered sufficiently developed for identification and ten larvae were 245 sampled from each. In October, where larvae were abundant, approximately 30 were sampled and if 246 fewer than this were seen, all larvae which could be captured were preserved.   Table 5. The development of Chaoborus from the initial egg rafts 287 introduced into each unit are presented for each replicate in Figure 3. Egg rafts added to the first 288 microcosms of replicates 1, 2, 4 and 5 failed to establish at the first attempt and replicates 1, 2 and 5 289 were re-initiated at intervals as fresh egg rafts became available. The re-initiated replicates 1 and 5 290 both progressed through to a fourth generation. These two replicates were found to contain both C. 298 299 Populations of Chaoborus in replicates 3 and 5 progressed through to a third and fourth generations 300 respectively. Larvae sampled from the first unit in July were found to be C. obscuripes, although 301 when re-sampled in October, both C. obscuripes and C. crystallinus were found to be present.
302 Generations 2 and 3, both sampled in October, were found to consist only of C. crystallinus. Replicate 303 6 also did not require re-initiation but as the second unit did not establish successfully, larvae were not 304 sampled from Unit 1 until October, in order to give the maximum opportunity for more egg 305 deposition to restart Unit 2. In practice, a second production of egg rafts did not occur in Unit 1 and 306 therefore, Unit 2 could not be re-started. Only six late-instar larvae remained in Unit 1 by the October 307 sampling and all were found to be C. obscuripes.

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309 Minimum egg-to-egg times for C. crystallinus are summarised in Table 4 and ranged from 14 days 310 (Replicate 5, Unit 1) to 56 days (Replicate 3, Unit 2). As only C. crystallinus was found in the 311 second, third and fourth generations of any replicate, it is not possible to draw any conclusions 312 regarding the egg-to-egg timings for C. obscuripes. The shortest generation time of 14 days occurred 313 when the water temperature was at its highest ( Fig. 3)