Sample Collection
Samples were collected between October 31st - December
10th, 2018 from Eucalyptus globulus Labill.
plantations around Hamilton, Victoria, Australia, owned and operated by
Australian Bluegum Plantations
(http://www.austgum.com.au/)
(Fig. 1) (SI Table 1 & 2). Plantations were chosen to ensure efficient
collecting. Another species of Eucalyptus was present as
volunteer plants but could not be identified to species due to an
absence of reproductive parts (Eucalyptus sp. 1). Beetle larvae
between the first and fourth instar were collected by hand and reared
gregariously in 750mL plastic takeaway containers, with all beetles
collected from a single tree reared in the same takeaway container.
Before pupation, beetle larvae were placed into 2cm x 2cm nylon pouches,
stapled shut, and isolated in small vials in anticipation ofEadya emergence to establish definitive host-parasitoid
associations following Davy et al. (2016). A total of 98 parasitoids
were reared from the beetle larvae.
For each beetle and parasitoid collected the GPS coordinates of the tree
were recorded. Upon reaching the final instar, the beetle host was
isolated before pupation when the parasitoid emerges to establish a
definitive parasitoid-host association. Upon emergence of a parasitoid,
what was left of the beetle host was immediately stored in 95% ethanol.
Parasitoid wasp larvae that emerged and successfully spun cocoons were
kept for 50 days, allowing for adult morphological characters to
develop, and then preserved in 95% ethanol. Those that failed to exit
the host or successfully spin a cocoon were immediately preserved in
95% ethanol. For each beetle larva collection event, three leaf samples
were also collected: an herbivore damaged leaf, an undamaged leaf of the
same age class as the damaged leaf (either flush (newly expanding) or
fully expanded glaucous foliage), and an undamaged leaf of the other age
class. Leaves were labeled accordingly and dried in individual plastic
sandwich bags with silica desiccating powder.