Mass Emergence of Scolytinid Beetles in a Seed Germination Experiment and Its Management

Uninvited Guests Mass emergence of Scolytinid beetles in a seed germination experiment and its management

Dirk HR Spennemann Kylie Kent Robert Cook

Uninvited Guests Mass emergence of Scolytinid beetles in a seed germination experiment and its management

Dirk HR Spennemann Kylie Kent Robert Cook

Albury December 2018

© 2018. All rights reserved by the author. The contents of this publication are copyright in all countries subscribing to the Berne Convention. No parts of this report may be reproduced in any form or by any means, electronic or mechanical, in existence or to be invented, including photocopying, recording or by any information storage and retrieval system, without the written permission of the authors, except where permitted by law.

Cover image: from left : Frass emerging from Phoenix canariensis seed; Coccotrypes dactyliperda seed from the side; view of a Coccotrypes dactyliperda breeding gallery © Dirk HR Spennemann 2018

Preferred citation of this Report Spennemann, Dirk HR, Kent, Kylie and Cook, Robert (2018) Uninvited guests: Mass emergence of Scolytinid beetles in a seed germination experiment and its management. Institute for Land, Water and Society Report nº 118. Albury, NSW: Institute for Land, Water and Society, Charles Sturt University. ISBN 978-1-86-467321-0

Contact Associate Professor Dirk HR Spennemann, MA, PhD, MICOMOS, APF Institute for Land, Water and Society, Charles Sturt University, PO Box 789, Albury NSW 2640, Australia. email: [email protected]

— ii — Table of Contents Introduction ...... 5 Experimental Design ...... 5 Experiment nº 2 ...... 7 Chronology of Emergence ...... 10 Containment ...... 10 Final disposal ...... 11 The species ...... 13 Life cycle ...... 13 Origin of beetles ...... 13 Implications ...... 23 Could the event have been foreseen? ...... 23 Could the event have been prevented? ...... 23 Did the containment work? ...... 25 Future Research ...... 28 Endnotes ...... 29 Acknowledgements ...... 30 Authorship contributions ...... 30 References ...... 30

— iii —

— iv — Introduction The principal author is engaged in a wider research project (‘Palms in the Anthropocene’) that aims to i) examine the history of introduction, marketing and distribution of Washingtonia spp. and Phoenix canariensis as horticultural plants, ii) review their role in urban landscaping, iii) compile the nature, role and range of volant and terrestrial dispersal vectors for these palms, iv) assess patterns of their establishment, naturalisation and adaptation to urban, peri-urban, and rural landscapes, and v) understand the role of these palms in the creation of novel ecosystems (Spennemann, 2018d, 2018e, 2018f, 2018g, 2018i, 2018j, 2018k, subm.-b, subm.-d, subm.-e). Distributed by the horticultural industry since the mid 1860s, and planted out in the open from the 1890s until the present, Canary Island date palms (Phoenix canariensis Wildpret) have been a common landscaping element favoured by those wishing to give their area an exotic feel (Spennemann, 2018a, 2019b; Zona, 2008). They have been employed as feature trees in private and public gardens as well as street trees lining avenues and boulevards. P canariensis has seen the most rapid and widespread uptake and today is distributed globally in warm temperate zones (Spennemann, 2018c). As the plants seed freely and their fruit (‘drupes’) are consumed by a number of vectors (Spennemann, subm.-e), they have become a weed species that is known to invade natural areas. The event discussed in this paper occurred during an experiment to assess the different germination potentials of the seeds of Canary Island date palm (Phoenix canariensis) drupes that had been ingested and subsequently spat out, regurgitated, or defecated by Grey-headed flying-foxes (Pteropus poliocephalus) (Spennemann, 2018e), Pied Currawong (Strepera graculina) (Spennemann, subm.-a), and Red Foxes (Vulpes vulpes) (Spennemann, 2018h, subm.-d) respectively.

Experimental Design The methodology used for the germination experiment followed that of Pimenta et al. (2010), with a regime of 16 hours of simulated daylight and 8 hours of darkness and with environmental temperatures fluctuating between 20°C during the ‘night’ and 30°C during the ‘day.’ The experiment had been set up in a climate controlled, window-less laboratory room of 2.6 x 2.8 m that had been designed as an animal holding room that forms part of a Physical Containment Level 2 (PC2) laboratory at Charles Sturt University, Albury–Wodonga campus. For the purposes of the germination experiment it was incidental that the room was attached to a PC2 laboratory, which at the time was not used for other experiments.1 Light was provided by three AquaOne® EcoGlo90 LED lights, each set up centrally above a plastic container. These lights primarily emit at the 430–490 nm spectrum (blue light) and 420– 780nm spectrum (white light, peaks at 460nm and 540–620nm). A stable ambient temperature was achieved by the room-specific air-conditioning system, which was manually adjusted twice a day. The temperature was set at the start of the simulated ‘day’ to 30°C (17:00–9:00) and let to cool off at the end of the ‘day’ with the night (9:00–17:00) a stable 20ºC. Given the comparatively small air space, both rise and fall of ambient temperature were relatively rapid. Actual room temperatures were logged with a TinyTag Plus 2 TGP-400 temperature and humidity logger (Figure 4–Figure 6). The experiment was carried out between 14 June and 27 July 2018. The samples were housed in three transparent plastic tubs with lids (34 l, 390 x 790 x 155mm), each filled with 13.33 l of evenly mixed planting substrate comprised of 50% peat moss and 50% Perlite. The surface was levelled and divided into a 3 x 6 grid using wooden skewers (Figure 1). Each cell was populated with nine seeds (3 x 3 pattern)(Figure 2). The seeds had been cleaned of adhering material (if any) and measured prior to the experiment (Spennemann & Pike, in prep.-b).

Figure 1. Layout of seeds in Tub B at commencement of experiment

Figure 2. Layout of seeds in Tub B Figure 3. Seeds marked as germinated in Tub B

No fungicides were applied in order to simulate natural conditions. The seeds were pressed into substrate, level with the surface, and with their operculum pointing upwards. The surface was then moistened with 1.35 l of standard tap water sprayed with a pressurised mist sprayer evenly across the surface of the tub. The lids were then replaced for the first 48hrs, after which they were removed. As the air conditioning vent was directly above the experimental set up, the surface was exposed to a continual light airflow. In consequence, on the evening of ‘day’ 7, the seed bed had dried out completely, and the experiment had to be adjusted and, essentially restarted. The surface of each tub was misted with 500ml of standard tap water and an additional 500ml were poured along the sides of the tub. Two days later, an additional 500ml were poured along the sides. From then on, the lids remained closed unless removed for a short time for the daily assessment of germination. The surface of each tub was evenly misted with 10ml of tap water 10 days and 13 days after restart, with an additional 20 ml per tub sprayed on days 16, 18, 22, 25, 28, and 30 after restart. These misting simulated morning dew and ensured that the surface of the exposed seeds did not fully dry out.

— 6 — 32

17 16/06/2018 17/06/2018 18/06/2018 19/06/2018

Figure 4. Example of diurnal variation. Temperature readings in five-minute intervals. The experimental set up comprised of three containers. Containers A and B, set up as replicates of each other (to the extent possible), housed seeds collected from Alma Park (NSW), while container C primarily housed seeds from Albury as well as additional seeds from Alma Park (for details see Spennemann & Pike, in prep.-a, to be subm.). Germinated seeds were marked with coloured pins (Figure 3) Experiment nº 2 During the ‘night’ of day nº 7 eleven sets of additional germination samples were placed into the same laboratory space. Each sample comprised of seeds placed between moistened sheets of paper kitchen tissue which itself was placed in a sealed plastic (Ziplock) bag. During the ‘night’ of day 8 a duplicate set of samples was added; in this set the seeds had been subjected to a 24-hr soak in tap water (Spennemann & Pike, to be subm.). Samples from this experiment allowed to assess the origin of the emerged beetles (see below).

— 7 —

18 12:00:00 AM 3:00:00 AM 6:00:00 AM 9:00:00 AM 12:00:00 PM 3:00:00 PM 6:00:00 PM 9:00:00 PM 12:00:00 AM Figure 5. Room temperature readings at 5-minute intervals, 14 June to 31 July 2018 (recorder nº 1; n=13,635).

18 12:00:00 AM 3:00:00 AM 6:00:00 AM 9:00:00 AM 12:00:00 PM 3:00:00 PM 6:00:00 PM 9:00:00 PM 12:00:00 AM Figure 6. Room temperature readings at 5-minute intervals, 15 June to 31 July 2018 (recorder nº 2; n=13,208)

— 8 —

a) b)

c) c)

e) f)

Figure 7. Patterns of germination and development of the radicle.

— 9 — Chronology of Emergence The germination of each seed was assessed every morning, which coincided with the end of sunlight hours of each experimental ‘day.’ At the end of ‘day’ nº 30 a small beetle was noted in Tub A. The next ‘day’ again a beetle (which may have been the same, or a different one) was noted in Tub A and collected, with a second specimen collected at the end of the following ‘day’ (32). At the end of ‘day’ nº 34 a major emergence of beetles had occurred, with numerous beetles trapped in condensation droplets on the walls of tubs A and B, and to lesser degree also on tub C. In total 72 beetles were collected. On day 35 a mass emergence took place. It appears that the emerging beetles became airborne and touched the lids of the tubs where they became trapped in condensation droplets. In excess of 128 beetles were counted trapped on the inside of the lid of tub A alone (Figure 8). A small number of beetles were noted on top of the tub lid as well as next to the tub. A similar situation was noted for tub B and to lesser degree on tub C. As the beetles were initially feared to be a Dermestid species that might have emerged from contaminated peat moss, containment measures were initiated during the ‘night’ of day nº 35 (see below) and the number of beetles dropped dramatically. Subsequently, the beetles were identified as belonging to the Curculionidae family, and then as one of the Scolytinids (see below). The origin of the beetles in the tubs cannot be ascertained unequivocally given the combination of seeds in the tub experiments (but see below, p. 13).

Figure 8. Coccotrypes dactyliperda trapped in condensation droplets on the inside of the lid of tub A.

Containment The plastic tubs had a tight-fitting, but not hermetically sealing lid with a concave lip that overlapped the convex edge of the tub. Any gap between the two was less than 1mm. After the mass emergence during day 35 (see above), containment measures were implemented. It was initially feared that the beetles might be

— 10 — Dermestes spp and thus would pose an immediate risk to the taxidermy teaching collections co-housed in the teaching and research laboratory. As an immediate containment measure the edge of the tub was covered with a 25mm-wide fabric- based medical tape (Leucoplast®) designed to strap muscles after sports strains. After application, the fabric of the tape was liberally soaked with camphor oil, a known repellent of moths and other insects (Perumal & Wheeler, 1997). The day after the commencement of containment, 2 specimens were found trapped in condensation droplets on the wall of tub A and one in a droplet in tub C (day 36). No specimens were noted on the following days (days 37–39). Two adults were found trapped in condensation droplets on the wall of tub C on day 40. On day 41 two adults were noted at tub A (one trapped in a droplet and, one outside the tub), as well as one trapped in a droplet in tub C. The containment appeared to be working, as the number of emerging beetles dropped dramatically the day after the application, with no beetles found to be crawling outside the three tubs. In parallel with the containment, efforts were made to identify the species. It was soon established that these were not Dermestid but Scolytinid beetles belonging to the bark and ambrosia beetle family. Until the exact species could be determined, the containment was continued in view of the ravages exacted on ornamental palms by the invasive shot-hole borer (Euwallacea fornicates), which inter-alia cause fusarium dieback in commercial and ornamental palms (e.g. Elena, 2005; Eskalen et al., 2013; Hodel, 2017) including palms in Australia (Summerell & Gunn, 2001; Summerell et al., 2006).

The specimens were eventually identified as Coccotrypes dactyliperda,2 which do not warrant containment to the same degree as Euwallacea. Final disposal The germination experiment nº 2 was terminated on 20 July 2018 and all sample residue was placed into a commercial freezer at –20°C for 5 days. The germination experiment nº 1 was terminated on 2 August 2018, with the three tubs likewise placed in to the commercial freezer (Table 6) for further analysis. It is common practice to use prolonged exposure at–20°C to effectively kill all insects. There are no reliable data of the ability of the beetle species involved (see below) to withstand exposure to freezing temperatures. Cooperband et al. (2016) found among another Scolytinid species (Euwallacea fornicatus) that four hours exposure to temperatures between -1ºC and -5ºC led to a mortality of 100% of larvae, 95.7% of pupae and 69.2% of adults. Longer exposure killed all adults. Thus one week of exposure at –20°C was deemed more than sufficient. After one week, some of the samples from germination experiment nº 2 which showed evidence of beetle attack were retained and the remainder discarded. Germination experiment nº 1 was retained in toto and remained in the freezer for future analysis.

— 11 —

Figure 9. Experimental set up of tubs A (front) to C (back). The lid of tub B has been removed to show containment measures (see Figure 10 for detail).

Figure 10. Camphor-oil soaked fabric-based medical tape covering the edge of the rim

— 12 — The species The beetles encountered were Coccotrypes dactyliperda (Fabricius, 1801), a spermatophagus beetle (ALA, 2018; Walker, 2006). The species is closely associated with the date palm (Phoenix dactylifera) complex and can be considered endemic to the Middle East and North Africa. Due to the trade in dates as fruit for consumption, and during the nineteenth century also due to the distribution of palm seeds for horticultural endeavours and as vegetable ivory for button manufacture, C. dactyliperda has become a true cosmopolitan species that can be found in most subtropical and temperate zones (Spennemann, 2019c). Life cycle As with other crypto-parasites, the entire life cycle of Coccotrypes dactyliperda can occur inside the seed (Blumberg & Kehat, 1982). After emergence from hibernation (see below), the first generation of female beetles to leave the brood chamber emerges during late June/early July (in the northern hemisphere) and attacks green drupes, causing the bulk of these to abscise one to two days later (Blumberg, 2008). The species also predates the date seeds of fallen dates, often after the pericarp has been eaten off by other animals, such as rodents. This continues until August, when a second generation emerges.3 Unmated Coccotrypes dactyliperda females are able to lay eggs that produce male offspring and then to mate with these to produce offspring of mixed sex (Gottlieb et al., 2009). Mated females start to lay eggs 3 days after inhabiting a new seed (Zchori-Fein, Borad, & Harari, 2006). Depending on the size of the seed multiple generations of beetles can hatch and reproduce inside the seed before emerging. A single date seed can concurrently house large numbers of eggs, larvae, pupae and imagines, with in excess of 80 individuals (females, males, pupae, larvae and ova) reported in the literature (Doumandji-Mitiche, 1977). The development period of a full life cycle from egg to adult for females is temperature and humidity dependent and has been reported as ranging from 22.2 days (at 28°C) to 49 days (at 20°C) (Blumberg & Kehat, 1982; Herfs, 1959). During the winter period imagines of female Coccotrypes dactyliperda enter a hibernation or dormancy period inside the seed in which they hatched. The commencement and termination of hibernation were linked to both temperature and photo-periods. Temperatures at or below 15ºC coupled with reduced photoperiods (short days) seem to initiate hibernation, while temperatures above 25ºC and longer photoperiods terminated hibernation (Boraei, 1994).

Origin of beetles Given the biological information compiled above, it is clear that at least some of the seeds had been infested with Coccotrypes dactyliperda. Given the time of year, it can be presumed that they had been hibernating and thus went undetected, but began to reproduce once exposed to favourable conditions. The question arises where the infested seeds came from. In view of the nature of the germination experiments (with seeds from various sources arranged in a grid pattern in the same tub, the samples in the tubs are non-conclusive. It is different for the seeds experiment nº 2. These were checked on a weekly basis after two weeks. At each inspection, all germinated seeds were removed. The final check occurred after four weeks (28 days) at which time the presence of adult beetles, as well as larvae was noted in some of the samples (Table 1). The emergence of beetles was confined to samples originating from palm nº 15 of the driveway at Glenalvon’, Alma Park (co-ords -35.583595, 146.784556)(Figure 16). The samples comprised of a grab sample collected under the palm and a sample of fresh and ripe drupes taken off an infructescence on 7 May 2018. The subsequent history of the samples is detailed in Table 6.

— 13 —

Figure 11. Coccotrypes dactyliperda. Total length ca 2mm.4

— 14 —

Figure 12. An infested seed of sample ATF2. Right: access hole to the brood gallery

Figure 13. Longitudinal cross-section of an infested seed of sample ATF2. The seed contained 87 Coccotrypus dactyliperda in various stages of development.

— 15 —

Figure 14. Close-up of one of the galleries. The operculum with the germinated shoot is at the left, the access to the brood gallery at right.

a b c d e

1 mm

Figure 15. Development stages of Coccotrypus dactyliperda (all retrieved from seed shown in Figure 12). a–larva (third instar), b–pupa, c–imago emerging from pupa, d–female, e–male.

— 16 — Table 1. Emergence of Coccotrypes dactyliperda beetles after 28 days in the samples of the plastic bag germination experiment Sample Sample Code Untreated Soaked 24hrs Albury 708 Forrest Hill Ave, grab sample ABX FH 4 — — 708 Forrest Hill Ave, fresh drupes, defleshed ABX FH 5 — — Currawong regurgitated ABX FC1 — — Alma Park Church Currawong regurgitated APS 25 — — Currawong regurgitated, dark seeds (old) APS 27 Dark — — Currawong regurgitated, light seeds (recent) APS 27 Light — — Alma Park ‘Glenalvon’ palm nº 15 fresh drupes, defleshed, Khalal stage ATF 1 — 5 fresh drupes, defleshed, Rutab stage ATF 2 2 5 grab sample under palm, light seeds (recent) AGR 1 1 13 grab sample under palm, Rutab stage AGR 2 6 7 grab sample under palm, dark seeds (old) AGR 3 larvae 3

Table 2. Appearance of frass at various seed samples Sample 30/06/2018 1/07/2018 2/07/2018 4/07/2018 Total AGR 1 7 2 2 11 ATF2 4 1 1 1 7 ATF 1 3 3 1 2 9 AGR 3 4 1 5 AGR 2 1 1 2 1 5 APF 10 1 1 2 4 APS 22 1 1 2 APV 1 1 1 2 APS 16 1 1 APS 7 1 1 ABX FH 3 1 1 ABX FH 1 1 1 2 APS 23 1 1 APS 25 5 5 ABX 708 Grab 3 3 ABX FC 1 4 4 APV 14 4 4 APS 17 3 3 APS 18 2 2 APS 27 D 2 2 APS 27 L 2 2 APS 9 2 2 APV 21 2 2 APF 2 1 1 APF 3 1 1 APF 8 1 1 APS 12 1 1 APS 15 1 1 APS 19 1 1 APS 2 1 1 APS 20 1 1 APS 24 1 1 APS 26 1 1 APS 4 1 1 APS 5 1 1 APV 12 1 1 Grand Total 23 9 6 55 94

— 17 — Table 3. Appearance of frass at various seed samples by class of seeds and dates Sample 30/06/2018 1/07/2018 2/07/2018 4/07/2018 Total L15 Grab Clean 7 2 2 11 L15 Grab Black 4 1 5 L15 Grab Brown 1 1 2 1 5 L15 Fresh Brown 4 1 1 1 7 L15 Fresh Orange 3 3 1 2 9 Fox 2 1 9 12 Flying-Fox 4 4 Currawong Light 3 1 24 28 Currawong Black 2 2 ABX currawong 1 1 5 7 ABX 708 grab 3 3 Albury Fresh drupes 0 Grand Total 23 9 6 55 94 Table 4. Appearance of frass at various seed samples by class of seeds. Average Time Sample Average StdDev Count L15 Grab Black 8.80 1.79 5 L15 Grab Clean 8.91 1.58 11 L15 Grab Brown 9.80 1.48 5 L15 Fresh Brown 9.00 1.53 7 L15 Fresh Orange 9.44 1.59 9 Fox 11.08 1.68 12 Flying-Fox 12.00 0.00 4 Currawong Light 11.50 1.29 28 Currawong Black 12.00 0.00 2 ABX currawong 11.29 1.25 7 ABX 708 grab sample 12.00 0.00 3 Albury Fresh drupes Grand Total 10.55 1.78 93 It is highly probably that that the infestation of the fractions of the grab samples (AGR1-AGR3) occurred from imagines in some of the seeds. After collection, the samples were stored at room temperature (15–18ºC) which would result in delayed maturation and/or prolonged hibernation. The samples were split into the three fractions, 36 days after collection (Table 6). By the time the seeds were planted, 38 days had passed. If beetles had hatched in the ziplock bag containing the grab sample, the specimens went unnoticed. Any frass from the establishment of new galleries would have been discounted among the other detritus. It must be assumed that seeds with galleries were planted in both the tubs and the bag experiment. On day 8 of the germination experiment as restarted (day 16 of original experiment, day 54 after sample collection) some seeds in tubs A and B were noted to exhibit what appeared to be a granular ‘growth’ (Figure 19a) emerging from the underside of the seeds. Other seeds exhibited this over the following days. The same was noted after two weeks in some of the bag samples (Figure 19b). Only sample sets that later exhibited mature beetles also showed evidence of this granular ‘growth’ (Table 5). In hindsight, this granular ‘growth’ appears to be frass from beetles establishing new galleries. Using the evidence of frass as the starting point of the pre-oviposition period, we can assume that oviposition might have occurred two or three days after the evidence of frass. This implies that the beetle emerged from hibernation within a week of the commencement of the warm temperature regime.

— 18 — Table 5. Frass noted on samples of the plastic bag germination experiment (at first inspection after two weeks) Sample Sample Code Untreated Soaked 24hrs Albury 708 Forrest Hill Ave, grab sample ABX FH 4 — — 708 Forrest Hill Ave, fresh drupes, defleshed ABX FH 5 — — Currawong regurgitated ABX FC1 — — Alma Park Church Currawong regurgitated APS 25 — — Currawong regurgitated, dark seeds (old) APS 27 Dark — — Currawong regurgitated, light seeds (recent) APS 27 Light — — Alma Park ‘Glenalvon’ palm nº 15 fresh drupes, defleshed, Khalal stage ATF 1 lots lots fresh drupes, defleshed, Rutab stage ATF 2 lots lots grab sample under palm, light seeds (recent) AGR 1 little some grab sample under palm, Rutab stage AGR 2 some some grab sample under palm, dark seeds (old) AGR 3 little some

Figure 16. Palm nº 15 Lieschke Driveway, Alma Park

— 19 — Table 6. Chronology of sample processing and observations Date Action 7 May 2018 grab sample collected (scooped up under palm, Lieschke driveway nº 15), kept in sealed ziplock bag in a cool room (temperature 13–18°C) fresh drupe sample of orange (ATF1) and brown drupes (ATF2) collected of inflorescence of the palm and kept in sealed ziplock bag 11 May 2018 25 orange (Khalal) (ATF-1) and 25 brown (rutab) drupes (ATF-2) (Figure 17) measured and weighed, then epicarp removed and seeds re-measured and weighed rest of fresh drupe samples remained in sealed ziplock bag 12 June 2018 grab sample (Figure 18) sorted into three fractions: light coloured seeds (AGR1), shrivelled up drupes (AGR2) and black coloured seeds (AGR 3). extraneous material discarded: bat ejecta, straw, gravel 13 June 2018 25 seeds of subsample AGR2 cleaned of epicarp and measured and weighed 25 seeds each of subsamples AGR1 and AGR 3 measured and weighed 14 June 2018 sub samples AGR1–AGR3 and ATF1– ATF2 planted in tubs A–C in incubation room 16hrs light at 30ºC, 8 hrs night at 20ºC 16 June 2018 remaining drupes of samples ATF1 and ATF 2 cleaned of epicarp and seeds measured and weighed remaining seed of samples AGR1-AGR 3 measured and weighed 21 June 2018 remaining samples of AGR1–AGR3 and ATF1– ATF2 split into two equal sets. Set 1 placed between moistened paper towels (in Ziplock bags) and placed in incubation room Set 2 soaked in tap water for 24 hours experiment #1 (tubs A–C) was restarted by moistening surfaces and seedbeds (see text) 22 June 2018 Set 2 placed between moistened paper towels (in Ziplock bags) and placed in incubation room 29 June 2018 Frass noted (but not understood as such) in tubs A, B and C 30 June 2018 Expanding frass noted (but not understood as such) in tubs A, B and C 1–4 July 2018 Frass noted (but not understood as such) in a large number of seeds in tubs A, B and C 14 July 2018 a beetle noted in Tub A 15 July 2018 a beetle collected in Tub A 16 July 2018 a beetle collected in Tub A 18 July 2018 major emergence of beetles in Tubs A–C 19 July 2018 mass emergence of beetles in Tubs A–C, more than 12 beetles on the lid in tub A alone containment measure initiated 20 July 2018 two beetles collected 21 July 2018 two beetles collected in tub A 22 July 2018 two beetles collected in tub A, one beetles in tub C 23 July 2018 no beetles seen 24 July 2018 two beetles collected in tub C 25 July 2018 two beetles collected in tub A, one beetles each in tubs B and C 26 July 2018 no beetles seen 27 July 2018 two beetles collected in tub A, three beetles each in tubs B and C 28 July 2018 no beetles seen 29 July 2018 15 beetles in tub A, 11 beetles in tub B and 5 beetles in tub C 30 July 2018 no beetles seen 31 July 2018 38 beetles in tub A, 12 beetles in tub B and 1 beetles in tub C 1 August 2018 no beetles seen 2 August 2018 22 beetles in tub A, 9 beetles in tub B and 1 beetles in tub C Termination of experiment #1a, all three tubs placed in freezer at –18°C to -20°C

— 20 — 25 mm

Figure 17. Phoenix canariensis drupes at Khalal stage (left) and Rutab stage (right). Sample harvested off palm nº 15 Lieschke Driveway, Alma Park.

Figure 18. Composition of the grab sample (palm nº 15) as collected (photo 12 June 2018)

— 21 —

a) Tub A Day 7 b) Tub A Day 10

c) Tub A Day 21 d) Bag ATF2 (no soak) Day 15

e) Bag ATF2 (no soak) Day 15 f) Bag ATF2 (no soak) Day 15

Figure 19. Frass among the seeds in the germination samples.

— 22 — Implications The unexpected mass emergence of the beetles raises three major questions which will be discussed in turn: i) could the event have been foreseen or ii) prevented and iii) did the containment work, or what other containment methods could have been used. Could the event have been foreseen? None of the experimental designs dealing with Phoenix canariensis seeds, and which were consulted for the study, comment on the presence or absence of insect attack (Chatty & Tissaoui, 1999; Pimenta et al., 2010; Singh & Bhargava, 2009). A number of sources on palm seed germination are likewise silent on the matter (Meerow & Broschat, 2017; Mifsud, 1996; Silverstein, 2005), or only refer to insect attack on seedlings (Donselman, 1982). Moreover, the presence of crypto-parasites such as Coccotrypes dactyliperda and Coccotrypes carpophagus as seed predators in Australia is under reported (Spennemann, in prep.). Consequently, the possibility of infestation by crypto-parasites was not recognised by the investigator (DHRS). In retrospect, however, in a specific search on the topic, three sources were located that refer to the possibility of insect infestation of mature seed collected from underneath source palms (Marcus & Banks, 1999; Rauch, 1998; Robinson, 2009). Marcus and Banks (1999), in their ‘Practical Guide to Germinating Palm Seeds’ generically note that [d]amaging insects such as seed-boring beetles may arrive with seeds. They may reduce germination and spread to other seed batches.” Could the event have been prevented? Two sources suggest the use of pesticides to pre-treat seed collected from underneath source palms by soaking in pesticide (Marcus & Banks, 1999; Robinson, 2009) or a 10% bleach solution (Robinson, 2009). Marcus and Banks (1999), recommend that “[t]o minimize these risks, seeds collected from the ground, whether in the wild or from cultivated plants, and seeds collected under unknown conditions should be soaked in a contact insecticide solution once the fruit pulp has been removed. The insecticide solution should be prepared at the same concentration you would use to spray for pests. … Soak larger, harder and less permeable seeds longer, from 20 to 45 minutes.” A short experiment was carried out to assess whether the soaking in insecticide would have been effective against Coccotrypes dactyliperda, in particular in view of the fact that the females tend to block the tunnel entrances with their bodies to protect larvae and pupae. None of the published sources specify the type of insecticide to be used. Based on a review of the literature on insecticide use in commercial date palm plantations in the Middle East5 it was decided to test both pyrethroid and an organophosphate insecticide.

A single Phoenix canariensis seed was placed into a 7.5ml vial where it was exposed to a single beetle,6 which was allowed to tunnel for eight days.7 The seeds were then extracted8 and, as cohorts of ten, were exposed for 20 minutes to one of the following three insecticide treatments: i) soak in 10% bleach; ii) soak in a synthetic pyrethroid (Cyhella®);9 and iii) soak in a phosphorothioate (organophosphate) insecticide (Fenitrothion 1000®).10 One sample of ten beetles was used as a control and exposed to 20 min soaking in tap water. At the end of the immersion treatment the seeds were rinsed in tap water, dried off and returned to single vials to observe the hatching success. Given that the first beetles tend to emerge from seeds after 17 to 25 days (Spennemann, 2019a), the experiment was terminated four weeks after the insecticide treatments (i.e. 36 days after start of tunnelling). At the termination of the experiment all seeds that had not yielded two emergences were cut open and inspected Given the life cycle of Coccotrypes dactyliperda comprised of an egg incubation period (6-10 days), larval stage (12–15 days) and pupal stage (3-4 days) (Spennemann, 2019a), the presence of pupae was interpreted as evidence of successful breeding.11

— 23 — After immersion, some beetles were found floating in the solution. They were removed and, where necessary, euthanised. Found floating were one beetle each in the Cyhella and Fenitrothion insecticides, two in bleach and three in tap water. All seeds were then dried and inserted into the vials. In addition, the actual depth of the penetration was not ascertained at the start of the immersion. Some had commenced tunnelling but abandoned them. That had not been noticed at the commencement of the experiment and only recognised when the seeds were cut open at the end of the experiment. This accounts for the smaller sample numbers in Table 7 Table 7. Resistance of Coccotrypes dactyliperda to insecticide treatment (inside seeds) Breeding Success Abandoned Mortality n 10% bleach 71.4 14.3 14.3 7 Cyhella 55.6 11.1 33.3 9 Fenitrothion 0.0 12.5 87.5 8 H2O 62.5 37.5 0.0 8

Table 8. Differences in mortality between treatments and controls. Statistically significant (p<0.05) difference from the control shown in bold italics.

H2O 10% bleach Cyhella Fenitrothion — !2= 1.142; !2= 3.108; !2= 11.664; H2O p= 0.2853 p= 0.0779 p= 0.0006 — !2= 0.711; !2= 7.502; 10% bleach p= 0.3991 p= 0.0062 — !2= 4.834; Cyhella p= 0.0279 Fenitrothion —

The rate of mortality ranged from 14.3% for the sample exposed to 10% bleach to 87.5% for the sample immersed in Fenitrothion (Table 7). As expected, the control sample immersed in tape water showed no mortality. Statistically, neither the treatment in 10% bleach nor the treatment with Cyhella showed significantly greater mortality than the control (Table 8). Only Fenitrothion exhibited a consistently significantly higher mortality than all other treatments. To assess the insecticide impact on unprotected beetles exposed to sprayed surfaces, beetles were forced to walk across filter paper wetted with insecticide (or control). Freshly bred beetles, grouped into four cohorts of eleven, were placed individually into the centre of a 11 cm diameter filter paper placed in centre of a 25 x 36 cm laboratory tray. Beetles were collected once they reached the edge of the tray and placed in a covered petri-dish. Mortality was recorded at 0, 15, 60, 120, 180 and 240 minutes after exposure. A final check occurred after 24 hours (1440 minutes). Any specimens found alive at the end of that period were euthanised in 90% alcohol. The mortality of Coccotrypes dactyliperda exposed to surfaces treated with standard insecticides is comparatively low (Table 9). As expected, the control showed 0% mortality. The greatest immediate mortality (36.4%) was observed with the 10% bleach solution, with all mortality occurring during the first hour. Beetles exposed to Cyhella showed no major mortality (9.1%) and likewise stabilised after the first hour. Fenitrothion on the other hand exhibited a delayed effect with mortality increasing at three hours after exposure (36.4% mortality after 24 hours). The observed differences between the control (tap water) and Fenitrothion became statistically significant (two proportion z-test; p<0.05) only well after the 4 hour mark (z=2.2111, p=0.0271). The second insecticide Cyhella, had no significant effect.

— 24 — The observed differences between the control and the treatment with 10% bleach, on the other hand, became statistically significant after one hour (z=2.2111, p=0.0271). Table 9. Survival of Coccotrypes dactyliperda exposed to walking over an insecticide-treated surface (filter paper) hours 0 0.25 1 2 3 4 24 tap water 11 11 11 11 11 11 11 Fenitrothion 11 11 11 11 10 8 7 Cyhella 11 11 10 10 10 10 10 10% bleach 11 8 7 7 7 7 7 Table 10. Survival of Coccotrypes dactyliperda immersed in liquid. Statistically significant (p<0.05) difference from the control shown in bold italics. minutes hours Treatment 0 15 30 45 60 3 9 12 24 48 tap water 10 — 10 10 10 10 9 10 10 10 10% bleach 10 9 7 9 9 7 — 0 0 — Note: tap water data in italics have been drawn from a parallel study (Spennemann, subm.-c) Did the containment work? One of the questions to be answered is whether the containment worked or whether the numbers declined purely due to the life cycle of the beetle. The chronology of events (Table 6) shows that the first emergence of a beetle was noted 30 days after the commencement of the experiment,12 with the mass emergence 34 and 35 days after commencement. At the time of camphor treatment, the number of emerging beetles declined but then rose again. The overall curve (setting aside he dip immediately after treatment, conforms with the natural emergence sequences observed in other experiments (Spennemann, 2018b), which suggests that the camphor treatment had only a time-limited effectiveness. An experiment was designed to formally assess whether fabric tape soaked with camphor oil would act as a deterrent. The experimental set up was comprised of a 200ml sample jar which acted as the main reservoir (jar A). Two same-sized sample containers (jars B and C), were connected to jar A by 96 mm long plastic vials (Ø 15 mm). The interior of both tubes was lined at one end with 25mm-wide fabric-based medical tape (Leucoplast®). Five pre-treated Phoenix canariensis seeds each were inserted to jars B and C. The pre-treatment comprised of a scarification of some of the surface with sand paper (Figure 21) to expose the albumen and immersion in 70% Ethanol (for 25 minutes). The seeds were immersed in ethanol because, when searching for new host locations, Coccotrypes dactyliperda reputedly reacts to kairomones from date seeds, especially alcohol-mediated fractions (El-Barbary, Donia, & Mostafa, 2002; Meisner, Weissenberg, Blumberg, & Ascher, 1985). A population of 10 beetles was placed into the central reservoir (A) and allowed to move freely.13 The tops of all three sample jars were covered with nylon gauze (nylon stocking) to allow for the air to ventilate (Figure 20).14 The experiment was kept at 21ºÇ for the first 6 hours and then moved a standard temperature of 28°C. At set intervals (1, 2, 3, 4.5, 6, 9, 12, 24, 30, 48, 72 hrs) the distribution of beetles in the three sample containers was counted.15 Three variations of the experiment were run concurrently, with three replicates each:

E-I: fabric tape of tube to container B soaked with camphor oil,16 the other tube left plain; E-II: fabric tape of both tubes soaked with camphor oil; and E-III: fabric tape of both tubes left plain. The results are shown in Table 11. At two hours some beetles had commenced tunnelling experiment E-III. Camphor oil unequivocally worked as a confinement agent for the first 12 hours. It is not clear from the experiment, however, whether camphor oil acted as an actual repellent, or whether it masked or

— 25 — overpowered the kairomones from date seeds and thus did not trigger directional movement. At 24 hours, however, some beetles had moved past the camphor barrier, presumably as some of the camphor oil had evaporated.

200ml container opening covered with nylon stocking to allow for air ventilation

Jar B Jar A Jar C ‘camphor’ ’reservoir’ ’plain’

strapping tape strapping tape 5 date seeds soaked with camphor 10 beetles unsoaked 5 date seeds

25ml vial tip cut off; on inside lined with strapping tape Figure 20. Experimental set-up for the containment experiment

Figure 21. Scarified date seeds prior to immersion in 70% Ethanol

— 26 — Table 11. Distribution of beetles over time (see Figure 20 for design) Tube to jars B and C without camphor Time Replicate 1 Replicate 2 Replicate 3 (hrs) Jar B Jar A Jar C Jar B Jar A Jar C Jar B Jar A Jar C start 0 10 0 0 10 0 0 10 0 1 5 3 2 0 8 2 0 9 1 2 5 2 3 2 5 3 0 8 2 3 5 2 3 2 5 3 2 4 4 4.5 6 0 4 2 5 3 2 4 4 6 4 2 4 3 3 4 9 4 2 4 3 3 4 12 5 0 5 3 2 5 24 2 1 7 30 2 1 7 48 2 1 7 72 2 1 7 Tube to jar B with camphor, tube to jar C without camphor Time Replicate 1 Replicate 2 Replicate 3 (hrs) Jar B Jar A Jar C Jar B Jar A Jar C Jar B Jar A Jar C start 0 10 0 0 10 0 0 10 0 1 0 3 7 0 10 0 0 10 0 2 0 2 8 0 8 2 0 8 2 3 0 1 9 0 5 5 0 6 4 4.5 0 0 10 0 4 6 0 6 4 6 0 4 6 0 6 4 9 0 4 6 0 4 6 12 0 4 6 0 3 7 24 1 0 9 1 2 7 30 1 3 6 48 1 2 7 72 1 2 7 7 Tube to jars B and C with camphor Time Replicate 1 Replicate 2 Replicate 3 (hrs) Jar B Jar A Jar C Jar B Jar A Jar C Jar B Jar A Jar C start 0 10 0 0 10 0 0 10 0 1 0 10 0 0 10 0 0 10 0 2 0 10 0 0 10 0 0 10 0 3 0 10 0 0 10 0 0 10 0 4.5 0 10 0 0 10 0 0 10 0 6 0 10 0 0 10 0 0 10 0 9 0 10 0 0 10 0 0 10 0 12 0 10 0 0 10 0 0 10 0 24 0 10 0 2 8 0 0 10 0 30 0 10 0 2 8 0 1 7 2 48 0 10 0 4 6 0 1 7 2 72 0 10 0 4 6 0 1 7 2

At the end of the experiment, after 72 hours, 23.3% of the beetles in experiment E-III had bypassed the camphor barriers.

— 27 — Future Research The mass emergence of Scolytinid beetles in the seed germination experiment, combined with the death of numerous seedlings that had been affected by beetle attack, opened up a new line of enquiry into the role and importance of Coccotrypes dactyliperda on the seedbank of ornamental palm species. The experimental set-up of long-day photoperiods (16L:8D) coupled with the high temperature regime of 30ºC days and 20ºC nights caused the imagines of female Coccotrypes dactyliperda to terminate their hibernation to commence oviposition and then to mate with their male offspring (if they were unfertilised). While majority of the seeds impacted by Coccotrypes dactyliperda failed to germinate, some of the seeds germinated although they had been penetrated. This suggests that the location of the penetration is of significance. Seeds where the embryo was affected by initial tunnelling failed to germinate, while the others did. The progressive consumption of the albumen by the beetle, however, implies that the emerging germ epicotyl has reduced energy, and thus is likely to wilt before it can form viable rootles and a photosynthetically active leaflet. More experimental work needs to be done to assess the extent of the impact. The emergence of Coccotrypes dactyliperda also led to a systematic review of the biology of the species (Spennemann, 2019a) and its global distribution (Spennemann, 2019c) as well as an assessment of the present and future status of the species in Australia (Spennemann, in prep.). A multi-factorial experiment, assessing food choices and emergence times is being carried out at the time of writing (Spennemann, 2018b).

— 28 — Endnotes

1. The choice of room was purely governed by the fact that this was one of two rooms that allowed for temperature control to be set independent of the rest of the laboratory.—The principal author had been inducted into WH&S and laboratory procedures as required; the co-authors are the laboratory manager (KK) and technician (RC) respectively. 2. Ken Walker (Museum Victoria, Melbourne) kindly pointed the principal author into the direction of Scolytinid beetles and then verified the identification of the species as Coccotrypes dactyliperda. Sixteen specimens were accessioned by the Melbourne Museum under inventory numbers COL 132345–COL 132360. 3. The rate of abscission varies, but when the infructescences are not protected from beetle attack by chemical or physical (bags) means, production losses usually range between 20 and 40% (Hussein, 1990; Kehat, Blumberg, & Greenberg, 1976). 4. Images taken with a hand-held Olympus TG-3 digital camera. 5. The bulk of literature on insecticide treatment of commercial date palms (Phoenix dactyliperda) focusses on the protection of fruits and palms and thus addressed the treatment of leaf and stems to prevent or treat infestation primarily by the Red Palm weevil (Rhynchophorus ferrugineus), but also other pests such as scale insects (Parlatoria blanchardii, and Phoenicococcus marlatti), and mealy bugs (Dysmicoccus brevipes) (Abd Rabou, Hussain, Elsharabasy, & Abouamerm, 2015; Aldawood et al., 2013; Azam & Razvi, 2001; El- Saeid & Al-Dosari, 2010; Khan, Azam, & Razvi, 2001). The insecticides used are systemic treatments in the form of pyrethroids, such as Cypermethrin (e.g. Totalene 45 EC), Deltarmethrin and azadirachtin- based products (e.g. Sunny Neem Oil 1500 EC)(Azam & Razvi, 2001; El-Saeid & Al-Dosari, 2010); and broad-scale applications, predominantly organophosphates, such as Carbosulfan (e.g. Marshall 25EC), Chlorpyrifos, Diazinon; Dichlorvos (e.g. Nogos 50EC, Dimethoate (e.g. Dimethoate 400EC); Fenitrothion (Sumithion 50 EC), and Trichlorphon (e.g. Trichlorphon 80 SP) (Abd Rabou et al., 2015; Azam & Razvi, 2001; El Ezaby, Khalifa, & El-Assal, 1998; Khan et al., 2001), as well as country specific insecticide ‘cocktails’ (e.g. Aldawood et al., 2013; El Ezaby et al., 1998; Obied, 1998). Phenylpyrazole powders (e.g. Fipronil) are occasionally used (Abd Rabou et al., 2015). Given the life cycle of Coccotrypes dactyliperda, however, the only inception point, at which such insecticides might be effective is when applied onto the green fruit. Indeed, Gerson and Applebaum (2015) note ‘[s]everal sprays with an organophosphate, applied during the season onto the green fruit may reduce the damage. However, as these pesticides harm the natural enemies of other date pests, they should be applied only as a last resort.” None of the literature on insecticide treatment of commercial date palms comments on the treatment of seed. 6. The beetles used here were drawn from a population bred a for multi-factorial experiment, assessing food choices and emergence times (Spennemann, 2018b). These beetles originated from the mass emergences discussed in this report.— In the light of previous experiences with penetrations of single seeds by multiple beetles (Spennemann, 2018b), single beetles were placed with a single seed each into small, numbered vials. 7. As mated females start to lay eggs between one and three days after inhabiting a new seed (Herfs, 1950; Zchori-Fein et al., 2006), the eight-day period was deemed sufficient for each beetle to establish a breeding gallery. 8. In total 40 vials were prepared, with a slight time lag in ‘inoculation’ (1-2 days) due to availability of beetles. In addition, some beetles refused to take to a seed and had to be replaced after 3 days. The minimum time of exposure was 7 days and the maximum time 10 days. To ensure that these time differences did not affect the outcomes in a systematic fashion, the 40 vials were randomly allocated to one of four cohorts of 10 (in MS Excel new numbers 1-40 allocated at random, then grouped A-D in sets of 10). These four cohorts were then randomly allocated to the experiments. Once exposed to the sample solution, the beetles were returned to the numbered vials, with the control beetles allocated numbers 1–10, the beetles in the bleach solution numbers 11 to 21, and so forth.

— 29 —

9. Cyhella (Zelam, 2014); active ingredient: Lambda-Cyhalothrin (NPIC, 2001).—The insecticide was diluted with tap water to a concentration 5ml/100l l as per manufacturer’s directions 10. Fenitrothion 1000 (Barmac, 2017); for biological effects see (NRA, 1999).—The insecticide was diluted with tap water to a concentration 1.2l/100l as per manufacturer’s directions. 11. The extracted seeds were placed in numbered vials to observe the emergence success and timing. At the time of exposure, the brood chamber would have comprised of the adult female as well as eggs and some larvae (for life cyle times see Spennemann, 2019a). The emergence of more than one adult beetle was taken as evidence of successful continued breeding as the second individual must have developed from eggs (or larvae) present at the time of immersion, or from subsequent oviposition events. 12. 68 days after collection of the grab sample. 13. See Endnote 6 for the origin of the beetles. 14. The experiments were conducted in the same laboratory room and under the same conditions as the initial experiment (16 hours light @ 30°C; 8 hours darkness @ 20°C) 15. The experiment was conducted 6–9 November 2018. 16. The procedure was to dip the end of the camphor-covered tube into the camphor oil to all for soak and even coverage.

Acknowledgements Ken Walker (Museum Victoria, Melbourne) kindly pointed the principal author into the direction of Scolytinid beetles and then verified the identification of the species as Coccotrypes dactyliperda. Mark Lavery (Mark's Spray Barn, Albury) kindly supplied the small quantities of insecticide needed for the beetle control experiment.

Authorship contributions Conceptualisation of the germination, containment and insecticide resistance experiments DHRS; technical implementation of the germination experiment DHRS, KK and RC; design of containment strategy RC and KK; execution RC; technical implementation of the containment experiment RC; report conceptualisation, research and writing DHRS; all images DHRS.

References Abd Rabou, E, Hussain, Elsharabasy, S.F., & Abouamerm, W.L. (2015). Estimation of Insecticide Residue in Date Palm Fruits after Controlling the Red Palm Weevil. International Journal of Food and Nutritional Sciences, 4(5), 27–31. Abd-Allah, F.F., & Tadros, A.W. (1994). Biological studies on the date stone beetle, Coccotrypes dactyliperda Fabricius (Coleoptera: Scolytidae). Alexandria Science Exchange, 15(1), 1-9. ALA. (2018). Atlas of Living Australia. Coccotrypes dactyliperda (Fabricius, 1801). Canberra: Commonwealth Scientific and Industrial Research Organisation. URL: https://bie.ala.org.au/species/urn:lsid:biodiversity.org.au:afd.taxon:9e3c3be9-fb9d-43b5-923d- 300739e93aa4. [Retrieved on Jul 20, 2018] Aldawood, A.N , Alsagan, F, Altuwariqi, H, Almuterl, A., & Rasool, K. . (2013). Red Palm Weevil Chemical Treatments in Date Palms in Saudi Arabia: Results of Extensive Experimentations. In Colloque Mediterraneen sur les Ravagelljrs des Palmiers NICE-16, 17 et 18 Janvier 2013 (pp. 1–5). Alfortville,: Association Française de Protection des Plantes. Azam, K.M., & Razvi, S.A. . (2001). Control of Red Palm Weevil, Rhynchophorus ferrugineus Oliver using Prophylactic Spraying of Date Palms and Trunk Injection. In A. A. Al-Badawy (Ed.), Proceedings of the Second International Conference on Date Palms (March 25-27, 2001) (pp. 211–215). Al-Ain, UAE: United Arab Emirates University. Barmac. (2017). Safety Data Sheet: Methograin® Fenitrothion 1000 Insecticide. Lidcombe: Barmac. Blumberg, D. (2008). Review: Date Palm Arthropod Pests and Their Management in Israel. Phytoparasitica, 36, 411–448.

— 30 — Blumberg, D., & Kehat, M. (1982). Biological studies of the date stone beetle, Coccotrypes dactyliperda. Phytoparasitica, 10, 73–78. Boraei, Hessien A. (1994). Effect of temperature and photoperiod on the dormancy of the date stone beetle, Coccotrypes dactyliperda. Journal of Agricultural Research, Tanta University, 20(1), 72-79. Chatty, Y, & Tissaoui, T. (1999). Effect of Temperature on Germination of Ornamental Palm Trees in Tunisia Proceedings of the Conference "II International Symposium on Ornamental Palms & other Monocots from the Tropics". vol. ISHS Acta Horticulturae 486. pp. 165–167. Cooperband, Miriam F., Stouthamer, Richard, Carrillo, Daniel, Eskalen, Akif, Thibault, T., Cosse,́ A.A., Castrillo, L.A., Vandenberg, J.D, & Rugman-Jones, Paul. (2016). Biology of two members of the Euwallacea fornicatus species complex (Coleoptera: Curculionidae: Scolytinae), recently invasive in the USA, reared on an ambrosia beetle artificial diet. Agricultural and forest entomology, 18, 223–237. Donselman, Henry. (1982). Palm Seed Germination Studies. Proceedings of the Florida State Horticultural Society, 95, 256–257. Doumandji-Mitiche, B. (1977). Les Pyrales des Dattes Stockees. Annales de l'Institut. National Agronomique, El Harrach, Alger, 7(1), 31–58. El Ezaby, F. A., Khalifa, A. Osman, & El-Assal, A. (1998). Integrated Pest Management for the Control of Red Palm Weevil Rhynchphorus Ferrugineus Oliv in the United Arab Emirates, Eastern Region, Al Ain Proceedings of the Conference "First International Conference on Date Palms", Al-Ain, United Arab Emirates, March 8-10, 1998. vol. United Arab Emirates University. pp. 269–281. El-Barbary, N.S. , Donia, A.R., & Mostafa, Ayman M. (2002). Effect of food preference and extracts of the date palm fruits and stones on the activity of the adult females of the date stone beetle, Coccotrypes dactyliperda Fabricus (Coleoptera, Scolytidae). Alexandria Journal of Agricultural Research, 47(2), 103–107. El-Saeid, Mohamed H., & Al-Dosari, Saleh A. (2010). Monitoring of pesticide residues in Riyadh dates by SFE, MSE, SFC, and GC techniques. Arabian Journal of Chemistry, 3, 179–186. Elena, K. (2005). Fusarium wilt of Phoenix canariensis: first report in Greece. Plant Pathology, 54(2), 244. Eskalen, Akif, Stouthamer, Richard, Lynch, Shannon Colleen, Rugman-Jones, Paul F, Twizeyimana, Mathias, Gonzalez, Alex, & Thibault, Tim. (2013). Host range of Fusarium dieback and its ambrosia beetle (Coleoptera: Scolytinae) vector in southern California. Plant Disease, 97(7), 938-951. Gerson, Uri, & Applebaum, Shalom. (2015). Plant Pests in the Middle East. Coccotrypes dactyliperda Fabricius. Last updated on Mar 4, 2015. Jerusalem: Department of Entomology, Hebrew University of Jerusalem. URL: [Retrieved on Aug 12, 2018] Gottlieb, D, Holzman, J, Lubin, Y, Bouskila, A, Kelley, S, & Harari, A. (2009). Mate availability contributes to maintain the mixed-mating system in a scolytid beetle. Journal of Evolutionary Biology, 22, 1526–1534. Herfs, Adolf. (1950). Studien an dem Steinnußborkenkäfer Coccotrypes tanganus Eggers. 2. Die Soziologie von Coccotrypes tanganus. Höfchen-Briefe für Wissenschaft und Praxis (Bayer Leverkusen), 3(1), 3–31. Herfs, Adolf. (1959). Über den Steinnussborkenkäfer Coccotrypes dactyliperda (F.). Anzeiger für Schädlingskunde, 32(1), 1–4. Hodel, Donald R. (2017). Polyphagous Shot Hole Borer and Fusarium Dieback Disease on Palms. PalmArbor(1), 1–17. Hussein, A.E. (1990). Date varieties and palm height in relation to infestation with date stone beetle, Coccotrypes dactyliperda F. (Coleoptera: Scolytidae). Annals of Agricultural Science, Moshtohor, 28, 2613– 2622. Kehat, M., Blumberg, D., & Greenberg, S. (1976). Fruit drop and damage in dates: the role of Coccotrypes dactyliperda F. & nitidulid beetles, and prevention by mechanical measures. Phytoparasitica, 4, 93–99. Khan, Akhtar Jamal, Azam, K.M., & Razvi, S.A. . (2001). Pesticide Residue Analysis of Date Palm Fruits by Gas Chromatography Mass Spectrophotometry. In A. A. Al-Badawy (Ed.), Proceedings of the Second International Conference on Date Palms (March 25-27, 2001) (pp. 211–215). Al-Ain, UAE: United Arab Emirates University. Marcus, Jeff, & Banks, Ken. (1999). A Practical Guide to Germinating Palm Seeds. Principes, 43(2), 56-59. Meerow, Alan W., & Broschat, Timothy K. . (2017). Palm Seed Germination (Vol. BUL274). Miami: Environmental Horticulture Department UF/IFAS Extension. Meisner, J., Weissenberg, M, Blumberg, D., & Ascher, K.R.S. (1985). Date palm fruit stone extracts as phagostimulants for the adult date stone beetle, Coccotrypes dactyliperda F. (Coleoptera: Scolytidae) / Dattelkern-Extrakte als Phagostimulantien für Imagines des Dattelkern-Borkenkäfers, Coccotrypes dactyliperda F. (Coleoptera: Scolytidae). Zeitschrift für Pflanzenkrankheiten und Pflanzenschutz, 92(3), 305-309. Mifsud, Stefan. (1996). Germinating Palm Seeds. Chamaerops, 23. NPIC. (2001). Lambda-cyhalothrin (Technical Fact Sheet) National Pesticide Information Center. Corvallis, Oregon.

— 31 — NRA. (1999). The NRA review of Fenitrothion. Existing Chemicals Review Program. Nº 2 Technical Report. Canberra: National Registration Authority for Agricultural and Veterinary Chemicals. Obied, Mustafa Mahmoud Abdella. (1998). Production and Protection of Date Palms in Sudan Proceedings of the Conference "First International Conference on Date Palms", Al-Ain, United Arab Emirates, March 8-10, 1998. vol. United Arab Emirates University. pp. 455-468. Perumal, P , & Wheeler, Michael. (1997). Traditional practices for the control of insects in India. Conservation Journal, 23, 8–10. Pimenta, Ricardo Soares, da Luz, Petterson Baptista , Pivetta, Kathia Fernandes Lopes, de Castro, Amanda, & Pizetta, Patricia Unger Cesar.́ (2010). Efeito da maturação e temperatura na germinação de sementes de Phoenix canariensis hort. ex Chabaud - Arecaceae. Revista Árvore, 34(1), 31–38. Rauch, Fred D. (1998). Palm seed germination. Horticultural Digest. Nº 107. Honololu: Department of Horticulture, University of Hawaii. Robinson, M. L. (2009). Cooperative Extension SP-02-09: Cultivated Palm Seed Germination. Reno: University of Nevada. Silverstein, Robin P. (2005). Germination of Native and Exotic Plant Seeds Dispersed by Coyotes (Canis Latrans) in Southern California. The Southwestern Naturalist, 50(4), 472–478. Singh, R.S. , & Bhargava, R. (2009). Effect Of Seed Treatments on Germination and Growth Behaviour in Date Palm (Phoenix Species) under hot arid Conditions. Journal of Tropical Forestry, 25(1–2), 42-48. Spennemann, Dirk H.R. (2018a). Canary Date Palms (Phoenix canariensis) in Australia: introduction and early dispersal. Palms, 62(4), 185–201. Spennemann, Dirk H.R. (2018b). An experimental evaluation of food preferences and associated hatching times of the date stone beetle, Coccotrypes dactyliperda (Scolytinae, Coleoptera). Institute for Land, Water and Society Report. Nº 120. Albury, NSW: Institute for Land, Water and Society, Charles Sturt University. Spennemann, Dirk H.R. (2018c). Geographical distribution of four key ornamental and production palm species Phoenix canariensis, P. dactylifera, Washingtonia filifera and W. robusta. Albury, NSW: Institute for Land, Water and Society, Charles Sturt University. Spennemann, Dirk H.R. (2018d). Nineteenth Century Depictions of the Canary Date Palm (Phoenix Canariensis). A Visual Dataset. Institute for Land, Water and Society Report. Nº 113. Albury, NSW: Institute for Land, Water and Society, Charles Sturt University. Spennemann, Dirk H.R. (2018e). Observations on the consumption and dispersal of Phoenix canariensis drupes by the Grey-headed flying fox (Pteropus poliocephalus). European Journal of Ecology, 4(1), 41–49. Spennemann, Dirk H.R. (2018f). Phoenix canariensis and Washingtonia robusta drupes consumed by the Pied Currawong (Strepera graculina). A photographic documentation. Albury, NSW: Institute for Land, Water and Society, Charles Sturt University. Spennemann, Dirk H.R. (2018g). Phoenix canariensis drupes consumed by the Grey-headed flying-fox (Pteropus poliocephalus). A photographic documentation. Albury, NSW: Institute for Land, Water and Society, Charles Sturt University. Spennemann, Dirk H.R. (2018h). Phoenix canariensis seed encountered in scats and ejecta collected at Alma Park. Albury, NSW: Institute for Land, Water and Society, Charles Sturt University. Spennemann, Dirk H.R. (2018i). Review of the vertebrate-mediated dispersal of the Date Palm, Phoenix dactylifera. Zoology in the Middle East, 64(4), 283-296. doi: 10.1080/09397140.2018.1514785 Spennemann, Dirk H.R. (2018j). Washingtonia robusta at the Kremur Street Wetlands, Albury, NSW. A photographic documentation. Albury, NSW: Institute for Land, Water and Society, Charles Sturt University. Spennemann, Dirk H.R. (2018k). Washingtonia robusta drupes consumed by the Grey-headed flying-fox (Pteropus poliocephalus). A photographic documentation. Albury, NSW: Institute for Land, Water and Society, Charles Sturt University. Spennemann, Dirk H.R. (2019a). Biology, ecology and distribution of the date stone beetle, Coccotrypes dactyliperda (Scolytinae, Coleoptera). Zoology in the Middle East, 65, [in press]. Spennemann, Dirk H.R. (2019b). Canary Date Palms (Phoenix canariensis) as ornamental plants. The first thirty years of the horticultural trade. Huntia, 17(2), [in press]. Spennemann, Dirk H.R. (2019c). Global distribution of the date stone beetle, Coccotrypes dactyliperda (Coleoptera: Curculionidae, Scolytinae). Journal of Insect Biodiversity and Systematics, 4(3), 203–226. Spennemann, Dirk H.R. (in prep.). The palm seed borer Coccotrypes dactyliperda: vectors and processes of its introduction to and dispersal within Australia. Austral Entomology, [projected journal]. Spennemann, Dirk H.R. (subm.-a). Consumption of Phoenix canariensis drupes by Pied Currawong (Strepera graculina). Australian Field Ornithology, [under review].

— 32 — Spennemann, Dirk H.R. (subm.-b). Palms fanning out. A review of vertebrate vectors responsible for the dispersal of Washingtonia filifera and W. robusta. t.b.a., [under review]. Spennemann, Dirk H.R. (subm.-c). Resilience of breeding Coccotrypes dactyliperda (Coleoptera, Curculionidae, Scolytinae) to ingestion by vertebrates. Physiological Entomology, [under review]. Spennemann, Dirk H.R. (subm.-d). The role of canids in the dispersal of commercial and ornamental palm species. Mammal Research, [under review]. Spennemann, Dirk H.R. (subm.-e). When Palms go AWOL. A review of vertebrate vectors responsible for the dispersal of the Canary Island date palm (Phoenix canariensis). XXXX, [under review]. Spennemann, Dirk H.R., & Pike, Melissa. (in prep.-a). Feral Phoenix Canariensis in an agricultural landscape: nature and patterns of of dispersal. Australian Journal of Botany, [projected journal]. Spennemann, Dirk H.R., & Pike, Melissa. (in prep.-b). Inter-specimen variability of drupes and seed among Phoenix canariensis palms in the Southern Riverina, Australia. tba, [projected journal]. Spennemann, Dirk H.R., & Pike, Melissa. (to be subm.). Rites of Passage: germination of regurgitated and defecated Phoenix canariensis seeds. Austral Ecology, [projected journal]. Summerell, B. A., & Gunn, L. V. (2001). First record of fusarium wilt of Phoenix canariensis in South Australia. Australasian Plant Pathology, 30(1), 75-75. doi:10.1071/AP01001 Summerell, B. A., Smith, D. I., Gunn, L. V., Smith, I. W., & Pascoe, I. G. (2006). Fusarium wilt of Phoenix canariensis in Victoria. Australasian Plant Pathology, 35(2), 289-290. doi:10.1071/AP06007 Walker, Ken. (2006). Pests and Diseases Image Library The palm seed borer Coccotrypes dactyliperda (Fabricius, 1801). Last updated on 30 Jan 2011. Canberra: Australian Government, Department of Agriculture and Water Resources. URL: http://www.padil.gov.au/pests-and-diseases/pest/main/135850. [Retrieved on Jul 20, 2018] Zchori-Fein, Einat, Borad, Chandresh, & Harari, Ally R. (2006). Oogenesis in the date stone beetle, Coccotrypes dactyliperda , depends on symbiotic bacteria. Physiological Entomology, 31(2), 164-169. doi:10.1111/j.1365- 3032.2006.00504.x Zelam. (2014). Cyhella Insecticide. Material Safety Data Sheet. New Plymouth, NZ: Zelam Pty Ltd. Zona, Scott. (2008). The horticultural history of the Canary Island Date Palm (Phoenix canariensis). Garden History, 36, 301–308.

— 33 —