JOURNAL OF ENTOMOLOGY EUROPEAN JOURNAL OF ENTOMOLOGY Seasonal changes in mycophagous insect communities

. The phenology of fungal fruiting has changed in the UK over the last 70 years, but whether the associated mycophagous insects are able to exploit ‘out of season’ fruit bodies is unknown. This study focused on whether fungal baits can be used as a proxy to examine changes in fungal fruiting on insect communities. Using Agaricus bisporus as a bait, mushrooms were placed into two separate woodlands monthly from November 2020 to July 2021. Megaselia ru ﬁ pes (Phoridae) and Bradysia spp. (Sciaridae) were reared from both wild fungi and fungal baits at different times, making them appropriate species to consider for possible host tracking. Various factors affect an insect’s ability to track a fungal host, these include host preference, season, period of fungal fruiting and age of mushroom. Increased fruiting of macrofungi in the future may bene ﬁ t generalist mycophagous insects, by providing enhanced temporal and spatial resource opportunities. Using fungal baits as a proxy for the effects of climate change on fungal fruiting should be bene ﬁ cial in uncovering the host preferences of mycophagous insects and may potentially indicate whether mycophagous insects can track fungal hosts across seasons.


INTRODUCTION
Many insects and fungi occupy the same or similar habitats, e.g., dead wood systems, the rhizosphere and areas containing rotting organic matter. Fungal and insect interactions are important as they refl ect ecological processes such as phenology, competition, and succession and facilitate understanding of population and community ecology (Hackman & Meinander, 1979;Väisänen, 1981;Hanski, 1989). Most research on fungal fruiting bodies and associated insects has occurred within boreal forests of Northern Europe, woodlands of the U.K. (Chandler, 1976;Jakovlev, 2011) and Japan (Tuno et al., 2019).
The close link between fungi and insects may be affected by climate change since growth and development of fungi and insects is highly dependent upon seasonality, temperature and humidity (Boddy et al., 2014;Cui et al., 2018). Climate change is strongly associated with changes in phenology of many fungi across the UK and more widely across Europe (Gange et al., 2007;Kauserud et al., 2008), as well as earlier fl ight times of insects and changes in range (Braschler & Hill, 2007). Much research on phytophagous insects shows a link between insect phenology and spatiotemporal availability of foodplants (Bridle et al., 2014), including the ability of insects to track their hosts and phenological mismatches between hosts and consumers (Zohner, 2018). However, whether such trends occur in mycophagous insects is unknown.

Field sites
Fungal baits were placed and collected from a mixed woodland in Heston, Hounslow, U.K. (51.488722, -0.363987) and a mixed woodland (Huntersdale) in Egham, Surrey, U.K. (51.41669, -0.57115). The mixed woodland in Heston is predominantly Ash (Fraxinus excelsior) with several mature Oaks (Quercus robur) and is situated between grassland and farmland, with a heavy clay-based soil. Huntersdale is dominated by Oak (Q. robur), Scots pine (Pinus sylvestris), Silver Birch (Betula pendula) and Beech (Fagus sylvatica) and situated on sandy soil along a slope (refer to Table S2 for tree species found in both locations). Locations were chosen since they are undisturbed, reducing the likelihood of samples being vandalised and containing mature woodland. The distance between the two locations is approximately 19 km, which reduces the chances of insects migrating from one area to the other as species such as Musca domestica have been found to fl y up to 7 km (Nazni et al., 2005).

Fungal baits
The cultivated mushrooms, A. bisporus, were supplied by Merryhill Mushrooms Ltd (Storrington, West Sussex, UK), grown in 17.5 × 17.5 × 17.5 cm containers on a medium of wheat straw, poultry and horse manure in a controlled-temperature room of 18°C, and took approximately three to four weeks to become viable for placement into the woodlands. Once mycelium was seen the mushrooms were watered (300 ml each spray) twice daily to maintain a wet, humid environment for effi cient growth. Once mushrooms had formed and had begun to sporulate (determined visually), they were placed in the woodlands. To calculate a general approximation of fruiting body volume, the water displacement method was carried out with the use of 40 mushrooms (20 old and 20 young): each mushroom was weighed individually and placed into a beaker of water, the volume of water displaced (cm 3 ) was collected and measured, then plotted against weight (grams) of the fruiting body. Results from linear regressions were interpolated to predict volumes for collected cultivated samples based on weight, separately for young and old mushrooms. Weights of mushrooms used for water displacement ranged between 2.34 g to 128.56 g.
To test whether age was an important factor in insect oviposition preference, four boxes of mushrooms were grown for each location, from March up until July. A two-week difference in growing time between young batches of mushrooms and old batches of mushrooms was maintained each month. Old mushrooms were typically shrunken and had lost a large amount of moisture as well as having a darker appearance. Overall, 739 cultivated mushrooms were placed into the woodland areas. Numbers of mushrooms collected per month along with total weight are given in Table S1.

Collection of wild mushrooms and fungal baits
Collection of wild fungi began on the 25th of October 2020 and continued monthly until July 2021. Fungal collections coincided with placement and removal of baits. The cultivated samples were left in the woodlands for fi ve days to allow suffi cient time for insects to locate them and for oviposition to occur (Ideo et al., 2008). Wild fungi were collected from an approximately a 100-m radius from the cultivated fungi samples, to determine whether insect communities reared from wild fungi were similar to those reared from cultivated samples. Host tracking of wild fungi by insects was not recorded or monitored as wild fungal fruiting did not occur each month and their emergence could not be accurately predicted. insect ranges may decrease with climate change, decreasing realised niche range (Halsch et al., 2021).
Although not uniform across all fungi, the effect of climate change may also impact mycophagous insects (Kauserud et al., 2008;Kauserud et al., 2010;Sato et al., 2012). Since many single season fungi now have two fruiting seasons (Gange et al., 2007) shifts in temporal or spatial distribution of fungi (Gange et al., 2018) will require mycophagous insects to adapt, either through developing a broader host preference, as seen in the butterfl y Aricia agestis (Bridle et al., 2014), through tracking the movement of the host from one area to another (Posledovich et al., 2018), or temporally by adapting growth rates to match fungal host emergence times. Increased temperatures, humidity and precipitation may suit some mycophagous insects: many Diptera require wet, humid microclimates for pupation, whereas many Mycetophilidae may be negatively impacted as they prefer drier conditions (Hutson et al., 1980). Increased temperatures may also result in faster decaying of fruiting bodies especially in ephemeral fruiting bodies such as Coprinus spp., which deliquesce within hours of fruiting (Kües, 2000). Since fruit body appearance is unpredictable (Moore et al., 2008), a standardised method of examining whether mycophagous insects might be able to take advantage of the increased spatial and temporal fruiting (Gange et al., 2018) is needed.
In order to simulate different fungal fruiting times, this study used monthly placement of commercially cultured Agaricus bisporus as a bait, providing insects with a resource across seasons as a proxy for the effects of climate change on fungal fruiting to determine whether mycophagous insects can track their fungal hosts. This is the fi rsttime such an approach has been used to investigate host tracking as a proxy for the effects of climate change, and as a preliminary study this may pave the way for future works considering a method to determine how mycophagous insects track fungal hosts in relation to climate change. Additionally, a native fungal bait over a long period provides a seasonal fungal presence and provides information about insect communities within the bait and a baseline comparison to insect communities of wild mushrooms.
Here, the fi rst hypothesis was that baits would be a good proxy for changes in fungal fruiting on insect communities and that insects which can utilise the baits will be able to track them through time. Secondly, age of mushroom bait was hypothesised to be a key factor in choice preference for mycophagous insects following physical and chemical changes of the fruiting body as it ages and decays. The main aims of this experiment were to understand whether insects could track their fungal hosts phenology over a period of 9 months (November 2020 to July 2021), to see whether tracking of fungal hosts was consistent between two locations and to understand whether age of mushroom is a key factor in mycophagous host preference and subsequent shaping of insect communities.
Identifi cation of wild fungi, to species level where possible, was based on physical observations (e.g., gills, sporocarp shape, substrate, spore prints), and verifi ed by experts. Fungal collection consisted mostly of Agaricales, but Bracket fungi (Polypores) were also collected when possible.

Insect rearing methods
All mushrooms collected were broken from the mycelium and weighed before being placed into emergence traps (NHBS Ltd, Totnes, Devon, UK). Emergence traps (https://www.nhbs.com/ insect-mosquito-breeder) contained single or multiple mushrooms dependent on the size of the mushroom. Irrespective of the number of mushrooms per emergence trap, the total approximate volume was known. As emergence traps contained varying numbers of mushrooms, count data of insects and which species was standardised against per unit volume (cm 3 ) of mushroom. Approximately 6-7 cm of John Innes no. 3 compost was placed into each trap to provide larvae with a habitat in which to pupate and misted daily to maintain humidity. All mushrooms collected between October and December 2020 were kept in a Modular Cold Room (PORKKA, Watford, UK), a temperature-controlled incubation unit at 20 degrees centigrade with a day-night cycle of 12 h for each. From January 2021 onwards the collected mushrooms were then transferred to a polytunnel to refl ect natural conditions with respect to light and temperature. Every two to three days the tubs were checked for insect emergence and lightly misted with a spray bottle with water when required.
All mushrooms collected from November to May were kept until mid-August 2021, as some eggs and larvae may have required a phase of diapause for extended periods of time before emerging. Mushrooms collected from June and July were kept until late September 2021. Insects were collected with the use of a pooter. Once emergence traps had been emptied of visible insects, the pooter was placed into the freezer for approximately 10 minutes to slow the movement of the insects substantially enough for straightforward placement into vials. They were then preserved in 70% ethanol for further analysis and identifi cation.

Identifi cation of Insects
References to Royal Entomological Society checklists, keys (Hutson et al., 1980;Chandler, 1998) and consultations with entomological experts (Peter Chandler, Henry Disney and members of the London Natural History Museum) aided insect identifi cation. A combination of wing venation and examination of genitalia were used for identifi cation (to species level where possible) to minimise error. Examination was performed with the use of compound and binocular microscopes followed by photography through a microscope lens with the use of a camera attachment and saved with computer software (Swift Imaging 3.0). Insect larvae which failed to pupate or become adults were not identifi ed due to diffi culties in accurate identifi cation. Ten individuals of Collembola were collected but omitted from analysis.

Statistical analyses
All statistical analyses were conducted in R studio Version 1.4.1717. These included a Two-Way ANOVA (R's Car package), a Negative Binomial GLM (R's MASS package), Poisson GLM, Non-Metric Multidimensional Scaling graph (NMDS) (R's vegan package), ANOSIM tests (R's vegan package) and Linear Regression analysis. Two-Way ANOVA's were implemented to compare means of insect abundances and means of insect species per unit volume of mushroom between months and location, months and age within the same location and between locations and age. When assumptions for a Two-Way ANOVA were not met, a GLM (Poisson or Negative Binomial) were implemented. Data used for NMDS were based on total abundances for each insect species per month for each location. NMDS were used to visually compare insect diversities between months, age of fruiting body, between locations and within locations (i.e., cultivated samples vs wild samples), and the Bray-Curtis dissimilarity method was used to test for dissimilarity between sites. ANOSIM tests indicated which factors were signifi cant and contributed most to the spread of data in NMDS graphs. Linear regressions were implemented to understand whether there was a relationship between volume of mushrooms and insect abundance as well as number of insect species.

Weather data
Weather temperatures for Heston were collected from Freemeteo (https://freemeteo.co.uk) which uses data from the Heathrow Weather Station (London, U.K.). Weather temperatures from Egham were collected from data recorded from a Weather Station located in Silwood Park Campus from Imperial College London, Buckhurst Road, Ascot, Berkshire SL5 7PY, U.K. Average temperatures were calculated based on minimum and maximum daily temperatures.

RESULTS
In total, 13,612 insects were reared between October 25 th , 2020, and July 22 nd , 2021, from cultivated (12,516 individuals) and wild fungal fruiting bodies (1,096 individuals) across both locations of Heston and Huntersdale. Cultivated samples from Heston produced 24 insect species from 18 different families while wild fungi produced 33 insect species from 19 families. Cultivated samples collected from Huntersdale produced 26 insect species from 21 different families while wild fungi produced 15 insect species from 8 different families. Graphs showing months indicate the time in which fungal baits were placed into each woodland and the associated number of insects and insect species produced from them. They do not show when insects emerged, as the latter process took place over extended and variable time scales. To see the complete list of fungal and insect species found in both locations please refer to Tables S3, S4 and S5.
Insect abundance collected from young, cultivated mushrooms was considerably higher in the summer months (June and July) compared to winter months (χ 2 = 72.85, df = 5, p < 0.001) (Fig. 1a), and this pattern was mirrored in numbers of insect species for both locations across the same period (χ 2 = 170.14, df = 5, p < 0.001) (Fig. 1b). Insect abundance and the number of insect species from young mushrooms were similar between the two woodlands although in Huntersdale fungal baits produced no insects from December to March, whereas in Heston there were no insects produced from January to March. A similar trend was found for insect abundance and insect species in old, cultivated mushrooms, with there being a higher insect abundance in summer months for both locations (χ 2 = 42.18, df = 2, p < 0.001) (Fig. 1c) and more species appearing in June and July in comparison to preceding months for Heston and Huntersdale (F 2,50 = 16.22, p < 0.001). Additionally, it appeared that there were more insect species using old, cultivated mushrooms in Heston compared to insect species using old, cultivated mushrooms in Huntersdale (F 1,50 = 5.18, p < 0.05) (Fig. 1d). When considering insect communities between each location, it appears that most insect families were present in both areas, irrespective of age of mushroom (Fig. 2a). Despite this fi nding, a small number of species were found in specifi c locations and age of sample, such as Culicoides spp. (Ceratopogonidae), Pediciidae (Diptera) and Erotylidae (Coleoptera) which were reared only from young, cultivated fruiting bodies in Huntersdale. Chalcidoidea (Hymenoptera) were only reared from old, cultivated samples collected from Huntersdale. Conversely Nemapogon cloacella (Lepidoptera: Tineidae) was only reared from cultivated samples in Heston. Interestingly, these families were not reared from any wild fungal fruiting bodies (Table  1). When considering season, it appears that insect communities change and differ considerably depending on the time of year (Stress = 0.09, R = 0.64, p < 0.001) (Fig. 2b).
Insect communities did not differ between locations within each month. Insect communities were dissimilar between cultivated mushrooms and wild mushrooms (Stress = 0.11, R = 0.42, p < 0.001), but not between locations (Fig. 2c).
M. rufi pes (Phoridae) and Bradysia spp. (Sciaridae) were the only two species which were collected throughout the period of sampling (Fig. 3a) using both wild fungi and fungal baits at differing times (Fig. 3a). The lowest average temperatures were between December and April and the highest temperatures were recorded in June and July (Fig.  3a).

DISCUSSION
This is the fi rst study in which fungal baits have been used over an extended period as a proxy for the potential effects of climate change on fungal fruiting and changes in associated mycophagous insect communities. It was not possible to fi nd conclusive evidence of host tracking by any of the insects reared from wild or cultivated fungal samples. Instead it was found that insect communities of A. bisporus were distinctly different to wild mushrooms in both locations, with many Mycetophilidae being collected from wild fungi (e.g., Exechia fusca and Mycetophila fungorum) and species of various insect families (e.g., Drosophilidae, Fanniidae and Muscidae) being collected from fungal baits (supplementary data Table S4 and * Ten individuals of Collembola were collected but omitted from analysis as focus was strictly upon insect communities.  (Komonen, 2003;Jakovlev, 2011;Põldmaa et al., 2015), but overall it seems Agaricus bisporus is not a suitable bait for acting as a proxy for monitoring changes in fungal fruiting on insect communities. Despite this, two species were successfully reared from wild fungi and fungal baits at differing times of the sampling period, namely M. rufi pes (Phoridae) and Bradysia spp. (Sciaridae). Suggestions of possible host tracking could be attributed to M. rufi pes which appeared in high numbers from fungal baits during April 2021 when there were very few wild fungal fruiting bodies in each location.
In terms of fungal age and host preference, it appears that there could be an element of resource partitioning between M. rufi pes and Bradysia spp. Despite using the same fungal host, M. rufi pes was not found in old fruiting bodies and was mostly reared from young specimens. Results for Bradysia spp. show presence in both old and young specimens, which suggests a successional component to fruiting bodies and the insect communities related to them.
The low abundance of insects and insect species from cultivated samples between the months of November and December 2020 in both locations is perhaps a result of the cold weather, which is detrimental to many insects as they can struggle to maintain core temperatures and mobility is heavily reduced (Teets & Denlinger, 2013). From wild fungi there were a few insect species that were able to emerge during the winter, namely T. fenestralis, Trichocera spp. and Boltiophila spp. Trichocera spp., are commonly known as Winter Crane fl ies and appear to be adapted for cold conditions (Hågvar & Krzeminska, 2007;Ci & Kang, 2021). As fungal fruiting predominantly occurs in the autumn it would be expected that the highest insect numbers would occur during this time, however the data suggests the opposite, the answer is likely due to the food preferences of the insects reared as there may be more insect species in summer which can exploit several food resources other than fungal material. This is evident in various species of Drosophilidae and Phoridae as well as other Dipteran families (Brown, 2001). High average temperatures during the months of June and July in comparison to previous months could have accelerated the rates of decomposition of organic matter in woodlands (Song et al., 2014), increasing the possible food resources available to a range of insect species; this is refl ected in the insect species and insect abundance reared from June and July samples, mainly consisting of Drosophillidae, Sphaeroceridae, Muscidae and Fanniidae, all of which contain species that are known to feed on a wide variety of decaying materials (Brown, 2001). Being able to consume fungal spores as well as rotting fungal material provides a perfect habitat for larvae of insects with such adaptability. M. rufi pes is a saprophage and can exploit a broad range of decaying materials including human cadavers (Disney, 2005), which suggests that their use of the fungal baits is likely to be opportunistic behaviour. M. rufi pes may be active at times which typically occur outside of fungal fruiting periods and can overwinter both as an adult and as a pupa (Eisenschmidt, 1958;Herbert & Braun, 1958), which may explain why it was absent in baits between January and early March.
In the case of M. rufi pes, specimens were reared from six species of wild fungi (Agaricus sylvaticus, Agaricus campestris, Calocybe gambosa, Coprinus micaceus, Macrolepiota rhacodes and Russula spp.) across this study. The use of Coprinus spp. (the 'ink-caps') was surprising as they usually deliquesce within a matter of hours, which suggests that this species can locate suitable food sources extremely quickly (Disney, 2005). Bradysia spp. were reared from fungal baits and three wild species (Auricularia auriculajudae, C. gambosa and Psathyrella spadiceogrisea). It is possible that many mycophagous insects are in the soil feeding on fungal mycelium and hyphae, and so it would be benefi cial to collect soil samples in future studies during periods of low insect abundance (Sawahata et al., 2002). It may be that 'generalist' insects which can utilise multiple fungal hosts are more likely to be capable of host tracking compared to 'specialist' insects, as suggested by our fi ndings with M. rufi pes and Bradysia spp.
Collection of wild fruiting bodies provided useful insights into how insect communities change over time. Mycetophilidae dominated in Huntersdale's wild mushrooms and yet they were rarely reared from fungal baits; this highlights the importance of fungal host preference for mycophagous insects. It has been suggested that fungal chemical compounds play a role in insects host preferences (Jakovlev, 2012;Leather et al., 2014) but season is also an important factor as a majority fungi tend to fruit at specifi c times, typically autumn and for fewer fungi, spring. The fact that fungal baits and wild mushrooms were present at the same time and produced differing insect communities suggests that fungal properties also shape associated mycophagous insect communities (Thorn et al., 2015).
Overall, this study has highlighted that fungal baits are useful for attracting and rearing a range of mycophagous insects which differ to wild fungi, but was inconclusive for whether mycophagous insects can track fungal hosts. Host tracking will likely be dependent on several different factors such as season, host preference, fungal fruiting period and age of fruiting body. The fact that M. rufi pes and Bradysia spp. were collected from wild fungi and fungal baits at differing times is interesting and requires further investigation. Although this study is inconclusive about whether insects track fungal hosts, it is important to note that it cannot be ruled out. Furthermore, the two species mentioned seem to display behaviours and preferences which would be suitable for host tracking (e.g., generalist saprophages which can exploit numerous food resources). The lack of Mycetophilids, which are strongly associated with fungi and thought to be predominantly mycophagous, collected from cultivated samples in this study, simply suggests that A. bisporus is unsuitable as a fungal host for this family, and so severely limited the number of insects that we could monitor and analyse for possible host tracking. We suggest that collaboration is needed between mycologists and entomologists to analyse the insect communities that occur in wild mushrooms fruiting 'out of season', to determine whether host tracking can occur. Periods of low insect activity also require attention as more information is needed on the general ecology of mycophagous insects. Fungal fruiting bodies should be seen as complex and diverse habitats for a range of organisms: their role as a food source is essential to many insects and is likely to play an important role in food webs especially in forest/woodland habitats. We suggest that naturally occurring, common species are used instead of A. bisporus. A. bisporus as a bait is simple and easy to grow but uncommon in woodland environments and seems to be avoided by most Mycetophilidae. Therefore, to test whether Mycetophilidae can track fungal hosts, an alternative cultivated fungus should be used which is naturally occurring and common in the wild, Pleurotus ostreatus may be a more suitable alternative for future studies. AUTHOR CONTRIBUTIONS. The study was designed by all authors and executed and analysed by R.B. All authors contributed to writing of the manuscript.

ACKNOWLEDGEMENTS.
Many thanks to C. Haokip, N. Morley and R. Prouse for their technical assistance. We are grateful to H. Disney, P. Chandler, B. Ferry, A. Polaszek and D. Sivell for their help with insect and fungal identifi cation and Merryhill Mushroms Ltd for provision of mushroom growing kits.