Appendix to Chapter 3, A non-destructive extraction method

Table A.1. The collection information for the pinned museum specimens of Megadromus antarcticus. All specimens were from the Lincoln University Entomological Research Museum.

Date collected Location Collector Sequence (yes/no)

12/09/1996 Prices Valley Marris, J. W. M. Yes

15/10/1996 Mt. Cavendish Emberson, R. M. Yes

15/10/1996 Mt Cavendish Emberson, R. M. Yes

26/11/1993 Killinchy Sivasubramaniam, W. Yes

30/11/1986 Arthur’s Pass Emberson, R. M & Syrett, P. No

30/11/1986 Arthur’s Pass Emberson, R. M & Syrett, P. No

01/01/1980 Orari Gorge Howe, P. Yes

09/11/1977 Coopers Knob Butcher, M. R. Yes

09/11/1977 Coopers Knob Butcher, M. R. Yes

15/08/1968 Lincoln College Kain, W. M. No

Table A.2. The list of all taq polymerases tried when amplifying the historical DNA extractions by PCR. Whether amplification was achieved is listed under the results.

Taq Name Manufacturer Description Results

Phusion taq Thermo Scientific High-fidelity No amplification

Amplitaq Gold Applied Biosystems High specificity, _{sensitivity and yield} Multiple amplifications; _{not specific to target}

Dream taq Thermo Scientific High sensitivity No amplification

i-taq iNtRON Biotechnologies Standard No amplification

Appendix B Captive rearing of Hadramphus tuberculatus

Hadramphus tuberculatus Pascoe 1877 was thought to be extinct after its last sighting in 1922 (Sherly 1989) but was rediscovered in 2004 (Young et al. 2008). Hadramphus tuberculatus is one of New Zealand’s most endangered invertebrate species and, as such, effective

conservation management is imperative. Captive rearing initiatives have been included in the species recovery plan. While the idea of captive breeding has promise, a great deal of

uncertainty regarding the best method for breeding large weevils, especially H. tuberculatus, remains a substantial obstacle. Several attempts have been made to rear large weevils, such as Lyperobius huttoni (Pawson et al. 2004), Hadramphus pittospori (Bennett 1987), and H. spinipennis (Schöps et al. 1999), with some success. All three studies managed to successfully breed weevils, although there was a high mortality rate in the larval stage with few surviving to adulthood. The adults seemed to be very short lived (Bennett 1987, Pawson et al. 2004). To this end we attempted to breed H. tuberculatus and L. huttoni in the insectary at Lincoln University.

Four H. tuberculatus were placed in a 60 cm by 60 cm by 60 cm insect tent with a single large planter containing one adult Aciphylla aurea, three A. aurea seedlings, and one A.

subflabellata (Figure B.1). Seven L. huttoni were placed in a similar enclosure containing one A. aurea, one A. dieffenbachii, and one A. subflabellata. The weevils were observed between 12:00 and 14:00 every two to three days between February 1 2012 and May 31 2012 by which time they were over-wintering beneath the soil.

Figure B.1. Enclosure used for H. tuberculatus.

Five sightings of H. tuberculatus were recorded, including the death of one specimen (Table B.1). Feeding was directly observed on A. subflabellata and feeding damage was observed on all Aciphylla (Figure B.2 & B.3). Two individuals were seen mating at the base of the A. subflabellata. The weevils presumably burrowed into the soil at the plant base when they could not be sighted as there were no obvious hiding places on the plants themselves. In the field, soil burrowing was typical behaviour of weevils when they were returned to their host- plant. A large A. aurea died in the enclosure seemingly from extensive root damage although no weevils, eggs, or larvae were found on the plant’s roots when it was extracted. All L. huttoni were accounted for at every observation until the 20th _{of April when they apparently}

went below the soil to over-winter. Foraging and feeding damage were observed on all Aciphylla plants. The weevils were prone to falling outside of the planter. The behaviour of quickly dropping off a plant due to moving shadows has been well documented in L. huttoni (Hunt 1996). Since the insectary is exposed to natural lighting and situated next to a cricket field, a cloud passing over and causing a shadow on a plant or sporting activity may cause the weevil to drop from the leaf of the plant. The design of the planter would not allow the weevil to crawl back into the planter. In contrast, H. tuberculatus never fell from its planter, although in the wild, sightings of the weevil falling to the ground from shadows has been documented. When spotted feeding in the enclosure, H. tuberculatus was never seen far from the centre of the plant and no feeding damage has been recorded from leaves that were hanging over the

edge of the planter. The difference in where L. huttoni and H. tuberculatus feed on the leaves may be why one is more prone to falling outside the planter.

Table B.1. H. tuberculatus sightings including information on behaviour and time of siting. Date Weevil ID Notes

8/02/2012 22 Climbing on the netting of the enclosure at 12:00

24/02/2012 27 Found dead at the base of an A. subflabellata at 14:00

26/02/2012 22 & 28 Mating at the base of A. subflabellata at 12:00

02/03/2012 28 Feeding on A. subflabellata at 13:00

Our findings suggest that H. tuberculatus is able to feed on more than just its host plant, Aciphylla aurea. The observed feeding on a different Aciphylla species is complimented by an observation of a captive H. tuberculatus feeding on carrot when given the choice between carrot and A. aurea. We also suggest that H. tuberculatus exhibits sporadic diurnal activity contrary to the prevailing opinion that H. tuberculatus is nocturnal. The cause of death in one specimen could not be determined, although age is a possibility. While positive mating was observed, no offspring were found. We do not plan to disturb the enclosure before spring as doing so may damage any larvae that may be present. Lyperobiushuttoni appeared to be more of a diurnal generalist than H. tuberculatus and may have a longer activity period before over- wintering. The warmer climate at a lower altitude may have influenced the duration of their activity. No mating was observed for this species and no young observed.

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