(Coleoptera: Cerambycidae) Larvae on a Modified Artificial Diet

General entomology 353

A method for rearing Arhopalus ferus (Mulsant) (Coleoptera: Cerambycidae) larvae on a modified artificial diet

A.M. Barrington, D.P. Logan and P.G. Connolly

The New Zealand Institute for Plant & Food Research Ltd, Private Bag 92169, Auckland 1142, New Zealand Corresponding author: [email protected]

Abstract Burnt pine longhorn (BPL), Arhopalus ferus (Mulsant) (Coleoptera: Cerambycidae), is an introduced species sometimes found in association with export logs and sawn timber. A rearing method was developed to produce larvae of a known age, number and quality for control trials. Growth of larvae from newly hatched to 5 weeks was measured on a standard cerambycid artificial diet and on modified diets. Replacing pine wood with pine bark sawdust increased survival at 5 weeks from 23% to 76% and mean weight from 9 to 21 mg. There were significant interactions between the influences of three factors (diet, period of rearing, initial larval density) on the weight of surviving larvae. Individual rearing was preferred for convenience and a standardised method was used to rear 8740 larvae for disinfestation trials. Establishment and survival to 6 weeks for these larvae was 97%.

Keywords burnt pine longhorn, Cerambycidae, forestry, disinfestation, survival, weight.

INTRODUCTION Burnt pine longhorn (BPL), Arhopalus ferus In 2012 the Stakeholders in Methyl Bromide (Mulsant) (Coleoptera: Cerambycidae:), is Reduction (STIMBR) requested a research an introduced xylophagous species that is a programme into options for improving the quarantine pest of export logs and sawn timber. current use patterns of fumigants and identifying Preferred hosts are standing dead and dying trees, new treatment technologies. As a consequence, and logs and sawn timber of Pinus spp. (Hosking the Insect Rearing group at Plant & Food & Bain 1977; Brockerhoff & Hosking 2001). Research was asked to establish a laboratory Occasionally BPL will also be associated with colony of BPL, and to develop a rearing method other tree species such as Norway spruce (Picea using artificial diet for the supply of larvae and alba), Douglas fir (Pseudotsuga menziesii) and other life stages. BPL has been reared on artificial larch (Larix decidua) (Brockerhoff & Hosking diets, but research was either preliminary (van 2001). As a consequence of the biosecurity risk Epenhuijsen et al. 2012) or there were no data of finding live BPL in association with harvested associated with the report of rearing (de Viedma logs, importing countries require measures to et al. 1985). Rearing of BPL was started in early mitigate this risk including methyl bromide 2013 using a cerambycid diet from Rogers et fumigation. al. (2002). Larvae were initially mass-reared

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© 2015 New Zealand Plant Protection Society (Inc.) www.nzpps.org Refer to http://www.nzpps.org/terms_of_use.html General entomology 354 to about 5 weeks old before separation into time of exposure for logs between tree harvest individual containers. While this method used and ship loading. An additional group was reared minimal labour and time, the number and size to 8 weeks to assess trends for larval combat and of larvae recovered from the diet after 5 weeks survival. Larvae were recorded as being involved in was highly variable and the establishment of larval combat when they had wounds as indicated larvae tended to be poor (ca <10%). The aim of by cuticular melanisation sometimes combined the research reported here was to improve the with flaccidity. The establishment of neonate method in order to reliably supply young larvae cerambycids on diet can be improved by adding for disinfestation research. some dried and powdered host material (Gardiner 1970). The standard diet (Diet 1, Table 1) was MATERIALS AND METHODS compared with one in which pine wood sawdust General method was replaced by pine bark sawdust (Diet 3, Table Adults (n=30-40) from the colony were 1) (G. Clare, Plant & Food Research, unpublished introduced to containers (4.25 litres) and folded data). The latter diet was included as Hosking & wax paper sections were added as a substrate for Hutcheson (1979) found that BPL larvae that had oviposition. Eggs were removed from oviposition fed on inner bark had a four-fold higher relative containers and kept in Petri dishes until hatch. growth rate than those fed sapwood. Neonate larvae were handled using a fine camel- In experiment 2 the standard diet, the bark hair brush. Egg-laying by adults, egg development sawdust diet and four other diets (Table 1) were and experiments with larvae were all carried out compared for their effect on larval survival in controlled temperature rooms at 20°C and a and weight at 5 weeks after inoculation. Newly 16 h photoperiod. hatched larvae (n=240/diet) were added to the The artificial diet described in Rogers et al. different diets in 24-well cell culture trays. Diets (2002), and variations of it, was used for rearing 1 and 2, and 4 and 5, were based on the standard BPL (standard diet, Table 1). Preparation of diet and bark sawdust diets respectively but with followed the method described by Rogers et al. reduced cellulose and no glucose to assess how (2002). Diet was poured into Petri dishes or relative changes to carbohydrate and protein trays while still hot. Before use, the surface of the affect larval growth. Samples (100 g) of the diet in Petri dishes was scored to facilitate larval standard diet, and diets 1, 3 and 4 were sent to burrowing and establishment. Blocks of diet Hill Laboratories for proximate analysis of their were cut into small cubes (ca 2 g), which were nutritional value. Diets 2 and 5 had included pressed into the wells of cell culture trays and reduced mould inhibitor as it can be associated scored using the flat edge of a weighing spatula. with high neonate mortality for some insects Diet in test tubes was also scored while the tubes (Wang et al. 2014). were still in racks. All diet was stored at 4°C until Further modification was made to improve needed, and left overnight in a sterile room to the rearing method. Reduced cellulose powder warm up and dry out before use. in Diets 4 and 5 lowered their viscosity, enabling them to be dispensed into individual tubes Development of a rearing method following Singh et al. (1983). Test-tubes with In experiment 1 the effect of diet, starting larval diet were then inoculated with a single larva, density and period of development on larval and plugged tightly with cotton wool. Test-tubes survival and growth was examined. Newly were superior to cell culture trays as they were hatched larvae (n=100 per treatment) were reared less labour-intensive to prepare with diet, and using three methods: one larva per cell each with contamination from mould was confined to ca 2 g of diet in 24-well cell culture trays, and 10 individual tubes rather than entire trays. Diet 5 or 20 larvae per 90 mm diameter × 25 mm high was selected for routine use as it had less mould Petri dish with ca 50–60 g of diet. Larvae were inhibitor than Diet 4 but was otherwise identical, reared to 6 weeks old to simulate a maximum and the results of experiment 2 suggested that

Table 1 Ingredients (shown in g or ml) for a range of artificial diets tested for their suitability to rear BPL from newly hatched larva to 8 weeks old. Values are amounts to make 1 kg of each diet. The standard diet was from Rogers et al. (2002) and other diets are modifications to that diet. Ingredient Standard diet Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Wheatgerm (g) 24.3 24.3 24.3 24.3 24.3 24.3 Wesson’s salt (g) 8.1 8.1 8.1 8.1 8.1 8.1 Agar (g) 32.5 32.5 32.5 32.5 32.5 32.5 Cellulose powder (g) 73 36.5 36.5 73 36.5 36.5 Pine wood sawdust (g) 48.7 48.7 48.7 Pine bark sawdust (g) 48.7 48.7 48.7 Yeast (g) 24.3 24.3 24.3 24.3 24.3 24.3 Casein (g) 24.3 24.3 24.3 24.3 24.3 24.3 Sucrose (g) 20.3 20.3 20.3 20.3 20.3 20.3 Glucose (g) 12.2 12.2 Water (ml) 697.9 770.0 770.0 697.9 770.0 770.0 Mould inhibitor1 (ml) 14.6 14.6 12.3 14.6 14.6 12.3 Codling moth vitamins2 (ml) 16.2 16.2 16.2 16.2 16.2 16.2 Ascorbic acid (g) 3.5 3.5 3.5 3.5 3.5 3.5 1From a stock solution containing 150 g methyl p-hydroxybenzoate, 200 g sorbic acid and 1700 ml 95% ethanol. 2Solution ingredients are given by Ashby et al (1985). replacement of cellulose by water (Table 1) had no diet and of developmental period on numbers of negative effects on growth and survival of larvae. dead and living larvae was tested by chi-square The success of the rearing method was assessed analysis of contingency tables. The effect of diet by recording the number of larvae surviving after on the mean weight of larvae for experiment 2 6 weeks at 20°C for 15 batches of 512-650 larvae was determined by a simple ANOVA with six inoculated for disinfestation research. levels of treatment (i.e. diet). Tukey’s HSDs were used to group the six levels. Statistical analyses For experiment 1 final weights of larvae RESULTS were analysed for the effect of density, diet There were significant interactions between the and developmental period by ANOVA. The influences of the three factors (diet, developmental model used those three factors and all of their period, initial density) on the weight of surviving interactions to establish whether the effects on larvae (P<0.01), the consequence of which is larval weight were independent of one another. that main effects of the three factors could not Since there was a highly significant three- be meaningfully ascertained. However, using the way interaction (P<0.01), all combinations groupings determined by the Tukey’s HSDs, a of those terms were consequently used as 12 direct comparison of marginal means showed levels of a single treatment in an alternative larvae on Diet 3 were clearly heavier than those ANOVA. Tukey’s Honest Significant Differences on standard diet for the same period and density (HSDs) calculated using R 3.1.2 (R Core Team) (Figure 1). The number of larvae provided with were used to group the 12 levels. A lattice plot Diet 3 (with pine bark) surviving to 6 or 8 weeks (Deepayan 2008) was used to visualise larval was superior to that of larvae fed standard diet weights for each combination of density, diet for all methods tested (overall means 77% versus and developmental period. The independence of 53.2%, P<0.01) (Table 2). Similar numbers of

Figure 1 Weights (mg) for recovered BPL larvae fed standard diet (Std) or Diet 3 (pine bark diet) for the first 6 or 8 weeks after hatch at 3 different initial densities (1, 10 or 20 larva per container). Means (solid circle within box plots) followed by the same letter are not different at P<0.05 (as determined by the HSD test).

larvae survived at 8 weeks (overall mean 62.3%) (average 9.3 g). The proximate analysis indicated compared with those counted at 6 weeks (67.8%) limited differences occurred in the nutritional (P>0.05) (Table 2). Few larvae appeared to be value for diets (Table 4). There was a reduction in killed by biting (5% and 10% in the pine bark total carbohydrate of 27 and 32% for Diets 1 and diet at 6 weeks for 10 and 20 larvae per Petri dish 4 respectively compared with the standard diet, respectively) as determined by observation of a but no difference in total protein between diets. combination of wounds and flaccidity. No larvae The standard diet and Diet 3 were very similar in were classed as killed by larval combat in the nutritional value. 8-week group. Of a total of 8740 larvae inoculated Survival after 5 weeks was superior (average individually into test-tubes with Diet 5, 2.4% 75.6%) for larvae reared on Diets 3–5 (with failed to establish or died before 6 weeks and pine bark) compared with the standard diet and a further 0.6% were discarded as being too Diets 1 and 2 (average 22.8%) (Table 3). There small for convenient handling (average weight were differences between the mean weights of 8 mg). This gave an average yield of suitable larvae reared on different diets (P<0.05). Average larvae for disinfestation trials of 97% (range weight for larvae on Diets 3–5 was greater 94.7-99.3%) at 6 weeks after inoculation of (average 20.8 mg) than for larvae on other diets diet with neonates.

Table 3 Survival (%) and mean weight (mg) of Table 2 Survival (%) for recovered BPL larvae BPL larvae reared on artificial diets for 5 weeks fed standard or Diet 3 (pine bark diet) for the at 20°C and 16:8 h light:dark. Mean weights with first 6 or 8 weeks after hatch at 3 different initial the same letter do not differ at P<0.05. SE is one densities (larva per container). standard error of the mean. Weeks Density Standard diet Diet 3 Diet Survival Mean weight ± SE 6 1 41 80 Standard 37.9 10.5±1.7 a 6 10 63 83 1 15.8 10.2±2.6 a 6 20 68 72 2 14.6 7.1±2.8 a 8 1 48 75 3 72.5 22.8±1.3 b 8 10 49 76 4 77.1 19.5±1.2 b 8 20 50 76 5 77.1 20.2±1.3 b

Table 4 Results of the Hill Laboratories proximate analysis for the nutritional value of selected diets showing percentages for each component. Diet component Standard Diet 1 Diet 3 Diet 4 Moisture 71 77 73 78 Ash 1.20 1.19 1.24 1.21 Total Nitrogen 0.72 0.73 0.73 0.73 Total Protein 4.5 4.6 4.6 4.6 Total Fat 0.7 0.6 0.7 0.8 Total Carbohydrate 23.0 16.6 21.0 15.6

DISCUSSION The aim of this research was to develop a reliable with 10 neonates. These larvae had access to an method for rearing large numbers of BPL on average 5–6 g of diet compared with ca 2 g of artificial diet for disinfestation research. After diet for larvae reared individually in wells of cell- initial experiments, the protocol enabled >8000 culture trays and 2.5–3 g for larvae inoculated larvae to be reared to 6 weeks of age and a size at 20 per Petri dish. Subsequent rearing in test- suitable for disinfestation trials with a mortality tubes confirmed that 2-3 g of pine bark diet per rate of less than 3%. The method involved individual was adequate for rearing BPL to 6 individually reared larvae provided with artificial weeks of age at 20°C. Survival declined for larvae diet modified from one that Rogers et al. (2002) reared for an extra 2 weeks on the standard diet used to rear the endemic cerambycid P. reticularis. but not clearly so for the pine bark diet. Van The basic nutritional requirements for normal Epenhuijsen et al. (2012) attributed poor survival insect growth and reproduction are well known in Petri dishes inoculated with 5–15 larvae to and are the basis of recipes for making artificial cannibalism. Larval combat may have been a diets. However, despite having an adequate cause of the decline on the standard diet but this nutritional basis, insect larvae may not establish was not detected in the present experiments. It and develop on some diets because feeding was considered that given ≥2 g diet per larva, stimulants are absent or feeding deterrents BPL could be reared communally to 6 weeks present (Nation 2008). Replacing pine wood of age without significant mortality associated sawdust with pine bark sawdust was associated with biting. While rearing in Petri dishes might with an improved survival and growth for be adequate for laboratory colonies, rearing in BPL and it is possible that components of pine individual tubes is better for supply. There is no bark are phagostimulants for neonates. It was possibility of damage through larval combat, observed that BPL neonates appeared to burrow larvae are easy to sort and count for supply, the into standard and pine bark diets equally, but tubes are convenient for shipment to other sites feeding was limited in the former. This result and the larvae can easily be extracted from the is consistent with Hosking & Bain (1977) and tubes for trials. Hosking & Hutcheson (1979), who found that Keeping BPL in culture may be useful for BPL larvae feed preferentially and grow relatively supplying eggs when field-collected adults are fast on inner bark. not available. Other cerambycid species have A constraint to rearing insects on artificial been reared from egg to adult on artificial diets to diet is the time and labour required to replace identify larvae (de Viedma et al. 1985), avoid field the rearing medium. Here an adequate quantity collection of immature stages (Garcia-Ruiz et al. of diet was necessary to allow 6 weeks of growth. 2012) and for supply of insects for biological The heaviest larvae after 6 and 8 weeks were and control studies (Keena 2005). Survival from generally those from Petri dishes inoculated egg to adult can be high with >80% achieved

© 2015 New Zealand Plant Protection Society (Inc.) www.nzpps.org Refer to http://www.nzpps.org/terms_of_use.html General entomology 358 for some species (Hatchett et al. 1973; Lee & Banno H, Yamagani A 1991. Life-cycle and Lo 1998). Many cerambycids have life-cycles larval survival rate of the redspotted of one or more years under natural conditions longicorn beetle, Eupromus ruber (Dalman) (Banno & Yamagani 1991; Keena & Moore 2010) (Coleoptera: Cerambycidae). Applied including BPL (Hosking & Bain 1977). Rearing Entomology and Zoology 26: 195-204. on artificial diet at constant temperatures can Brockerhoff EG, Hosking GP 2001. Arhopalus substantially accelerate development. Van tristis (F.) (Coleoptera: Cerambycidae) Epenhuijsen et al. (2012) reared a small number (=Arhopalus ferus (Mulsant)): Burnt pine of BPL from egg to adults on standard diet at longhorn beetle. Forest and Timber Insects in 20°C and found that development took 278 New Zealand 27: 1-8. days. Since that study more beetles have been Deepayan S 2008. Lattice: Multivariate Data reared, and development on standard diet was Visualization with R. Springer, New York. highly variable with some beetles emerging ISBN 978-0-387-75968-5. in less than 3 months and others in 2 years de Viedma MG, Notario A, Baragano JR 1985. (A. Barrington, unpublished data). Variable Laboratory rearing of lignicolous Coleoptera development is a challenge to managing a colony (Cerambycidae). Journal of Economic and has been observed in other cerambycids Entomology 78: 1149-1150. (Pershing & Linit 1989; Keena 2005). Part of Garcia-Ruiz E, Marco V, Perez-Moreno I 2012. the variability may be due to an inadequate diet. Laboratory rearing and life history of an For BPL, rearing entirely on a pine bark sawdust emerging grape pest, Xylotrechus arvicola diet instead of the standard diet may be an (Coleoptera: Cerambycidae). Bulletin of improvement. A second cause may be variable Entomological Research 102: 89-96. diapause incidence when rearing occurs under Gardiner LM 1970. Rearing wood-boring inappropriate photoperiod and temperature beetles (Cerambycidae) on artificial diet. The conditions (Shintani 2011). Canadian Entomologist 102: 113-117. In conclusion, the present experiments Hatchett JH, Jackson RD, Barry RM 1973. indicated that a diet with pine bark sawdust Rearing a weed cerambycid, Dectes texanus, was consistently superior to a diet with pine on an artificial medium, with notes on wood sawdust in terms of establishment of biology. Annals of the Entomological Society neonates and larval growth to 6 or 8 weeks at of America 66: 519-522. 20°C. A method derived these from experiments Hosking GP, Bain J 1977. Arhopalus ferus had negligible loss of larvae, minimal labour (Coleoptera: Cerambycidae); its biology requirements and met the need for a reliable in New Zealand. New Zealand Journal of supply of larvae for disinfestation trials. Forestry Science 7: 3-15. Hosking GP, Hutcheson JA 1979. Nutritional basis ACKNOWLEDGEMENTS for feeding zone preference of Arhopalus ferus Funding for this study was provided by the (Coleoptera: Cerambycidae). New Zealand Stakeholders in Methyl Bromide Reduction Journal of Forestry Science 9: 185-192. (STIMBR) project. We also thank Barbara Keena MA 2005. Pourable artificial diet Waddell and Dave Rogers who reviewed the for rearing Anoplophora glabripennis manuscript (Plant & Food Research). (Coleoptera: Cerambycidae) and methods to optimize larval survival and synchronize REFERENCES development. Annals of the Entomological Ashby MD, Singh P, Clare GK 1985. Cydia Society of America 98: 536-547. pomonella. In: Singh P, Moore RF ed. Keena MA, Moore PM 2010. Effects of Handbook of Insect Rearing. Elsevier Science temperature on Anoplophora glabripennis Publishers, Amsterdam. Pp. 237-248. (Coleoptera: Cerambycidae) larvae and pupae. Environmental Entomology 39: 1323-1335.

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