TOC o “1-3” h z u RESPONSE OF PHORACANTHA BEETLES (Coleoptera: Cerambycidae) TO SOME VOLATILE COMPOUNDS OF EUCALYPTUS TREE SPECIES IN ZAMBIA PAGEREF _Toc517887312 h 11.0 INTRODUCTION PAGEREF _Toc517887313 h 11.1 PROBLEM STATEMENT PAGEREF _Toc517887314 h 21.2 PROBLEM JUSTIFICATION PAGEREF _Toc517887315 h 31.3 MAIN OBJECTIVE PAGEREF _Toc517887316 h 41.4 SPECIFIC OBJECTIVES PAGEREF _Toc517887317 h 41.5 HYPOTHESIS PAGEREF _Toc517887318 h 41.6 SCOPE OF STUDY PAGEREF _Toc517887319 h 41.7 ASSUMPTIONS PAGEREF _Toc517887320 h 42.0 LITERATURE REVIEW PAGEREF _Toc517887321 h 53.0 METHODOLOGY PAGEREF _Toc517887322 h 63.1 PROCEDURE PAGEREF _Toc517887323 h 63.2 STUDY AREA PAGEREF _Toc517887324 h 73.3 SAMPLE SIZE PAGEREF _Toc517887325 h 73.4 TREATMENTS PAGEREF _Toc517887326 h 73.5EXPERIMENTAL DESIGN PAGEREF _Toc517887327 h 83.6 DATA COLLECTION PAGEREF _Toc517887328 h 83.6.1 DATA COLLECTION FORM PAGEREF _Toc517887329 h 93.7 DATA HANDLING AND ANALYSIS PAGEREF _Toc517887330 h 94.0 TIME FRAME PAGEREF _Toc517887331 h 105.0 BUDGET PAGEREF _Toc517887332 h 116.0 REFERENCES PAGEREF _Toc517887333 h 12
1.0 INTRODUCTIONPhoracantha recurva and Phoracantha semipunctata are both in the family Cerambycidae, the order Coleoptera and the genus Phoracantha. They have been identified to be serious borer pests of eucalyptus, CITATION Duf l 1033 (Duffy, 1963), mostly those planted outside their natural range. An adult P. semipunctata has length of about 22-28mm. The body is shiny dark reddish-brown; a yellowish band reaches across the upper half of the elytra (the hardened forewings) with one yellow spot and a prominent spine at the end. The antennae of the females are nearly the same length as the body; those of the males are longer and heavier, with spines on segments 3-8. While an adult P. recurva’s head, pronotum and ventrites are reddish-brown to dark reddish-brown. Its legs and antennae are yellowish-brown to reddish-brown. The antennae are moderately longer than the body in the females and 1.6 times as long as the body in the males. There are very long, dense, golden hairs immerging from the underside of each body segment. The elytra are pale yellowish-brown with dark reddish-brown to blackish-brown markings. There is a slim, incomplete zigzag band towards the center of the elytra, and in most cases this is reduced to a small spot on each elytron. The fascia towards the tip of the elytra is wide at the suture and narrow at the margin. The female is 19.5-29.2 mm long and the male is 15-28.8 mm long, CITATION Han93 l 1033 (Hanks, 1993).

In Australia they are known to be minor pests attacking damaged, stressed or newly felled trees, CITATION Too35 l 1033 (Tooke, 1935) but they have become established in many temperate and tropical regions worldwide where they have been known to kill even healthy trees. Both species occur wherever Eucalyptus species grow, CITATION Fox80 l 1033 (Fox, 1980). They are a global pest of eucalyptus trees.

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Field surveys in southern California show that P. semipunctata and recurva are present from late April until October, CITATION Han93 l 1033 (Hanks, 1993). These insects use blends of volatiles released from eucalypts to select stressed host trees for feeding and oviposition. These volatile organic compounds emitted by trees mediate a variety of ecological interactions between trees and the insects. The ecological factors interact in complex ways to regulate the beetle populations, CITATION Pai01 l 1033 (Paine, 2001). The volatile compounds involved in the selection and rejection of host trees have been earlier studied and identified, CITATION Bar00 l 1033 (Barata, 2000). These volatile compounds play vital roles in triggering beetle behaviors such as attraction to tree for nectar and pollen feeding and oviposition, CITATION Bru11 l 1033 (Bruce TJA, 2011). The beetles respond to the host tree volatiles and yet details of how these insects perceive the complex profile of volatiles from different potential eucalyptus host tree species have not been studied in Zambia. The beetles are important pests of eucalyptus but may differ in their preferences for different species of this host.

Therefore, in order to help contribute to the knowledge on how to protect and conserve eucalyptus trees and wood, the study of determining the response of Phoracantha beetles (Coleoptera: Cerambycidae) to volatile compounds by using an olfactometer and also a field experiment will be conducted.
The findings of this topic will help determine the effective response of Phoracantha larvae to Eucalyptus volatile compounds and possibly identify new microorganisms associated with Phoracantha beetles in commercial plantations; in turn this will ensure the protection and at the same time it will promote a better economic stability for plantations in Zambia by utilizing healthy Eucalyptus trees. Hence this research is worth conducting.

1.1 PROBLEM STATEMENTThe two species of cerembycid beetles Phoracantha semipunctata (F) and Phoracantha recurva (N) are the most harmful xylophagous pests of Eucalyptus species that have been accidentally introduced from Australia into Zambia and most regions of the world in which their hosts have been planted. The beetles cause extensive tree mortality in plantations and woodlots plantings and are of serious economic concern. The insects attack mainly freshly cut logs or drought weakened trees which rapidly succumb and die, CITATION Men84 l 1033 (Mendel, 1984.). They continue to cause serious damages mainly after drought period. The insects may occasionally attack healthy and vigorous trees when population levels are high. The larvae can destroy virtually the entire cambium layer of young trees resulting in rapid death of the trees and in older trees may not destroy the complete cambium but can weaken the tree and expose it to further attack by later generations of the beetles CITATION Dri75 l 1033 (Drinkwater, 1975.). The serious infestations noted in Zambia in the 1970’s when beetle populations were recognized, a problem caused by multiple factors such as the absence of natural enemies for the beetles. The insects are very destructive to Eucalyptus trees infesting a major number of species grown in Zambia killing the trees and causing considerable losses. The insects have been reported in all parts of the country wherever eucalypts are planted.

Therefore, this research topic aims at determining the effective response of Phoracantha larvae to Eucalyptus volatile compounds and possibly identify new microorganisms associated with Phoracantha beetles in commercial plantations and this will be done by determining the response of Phoracantha beetles (Coleoptera: Cerambycidae) to volatile compounds produced by Eucalyptus trees.

1.2 PROBLEM JUSTIFICATIONPhoracantha beetles are a major pest of Eucalyptus species and the most economically damaging insects causing extensive tree losses in Zambia. They are well known plantation pests of drought stressed host trees which cause considerable damage under drought conditions. Hence as the droughts are expected to increase with climate changes in the country, outbreaks of the beetles are also expected to become frequent and intense. For example, in Southern parts of Africa, plantation forests are already experiencing significant shifts in annual rainfall and temperature subsequently increasing tree water stress and this may result in large amounts of tree mortality.

Presently, there is still a dramatic increase in the damage of Eucalyptus plantation caused by Phoracantha beetles and there has been no successful control measure. The increasingly loss of trees due to insect attacks is threatening the sustainability of the Eucalyptus species, timber and pole industry in Zambia. There is little doubt that these beetles are set to challenge eucalypt plantation forestry development in the country more than ever before. In order to sustain profitable business based on eucalypt plantation, the Forestry Department, Zambia Forests and Forestry Industries Corporation Limited (ZAFFICO) and Government will need to invest substantially in technologies enabling management of these pests.

This study will help document the diversity of microbial assemblage and possibly identify new microorganisms associated with Phoracantha beetles in commercial plantations. In light of the environmental problems caused by beetle damage it is necessary to integrate pest management in the overall tree improvement and breeding program. Thus, there is a need for insect control and minimal environmental impact. Furthermore, future integrated pest management programs of the beetles must integrate approaches targeting the pest into strategies for managing the pest complex; such an approach will require detailed knowledge of the pest interaction with other members of the community. The information generated from this study will be critical for future integrative efforts.

1.3 MAIN OBJECTIVETo ascertain the effect of volatile compounds produced by Eucalyptus trees on the effective response of Phoracantha beetles.

Collect leaf and bark samples of four commercially grown Eucalyptus trees.

Extract the Eucalyptus essential oils from the laboratory.

Analyze the data and make recommendations.

1.5 HYPOTHESISThe null hypothesis states that Phoracantha larvae to be examined will respond in the same way to all the different treatments.

And the alternative hypothesis is that at least one or more responses of Phoracantha larvae to treatments will be different from the rest.
1.6 SCOPE OF STUDYThe laboratory bioassay experiment will be confined to analyze the effect of the three different treatments on the attractive response of Phoracantha larvae. The treatments include: Volatile compounds produced by Eucalyptus leaves and bark for different species and the control.

The field experiment will analyze the response of the beetles to the different Eucalyptus species.
1.7 ASSUMPTIONSThere will be no disturbance to the field traps.
All the beetles will respond to the semiochemicals in the same manner.

2.0 LITERATURE REVIEWPhoracantha semipunctata and recurva attack Eucalyptus and minor species that are related in genera CITATION Duf l 1033 (Duffy, 1963). Research shows that wherever Eucalyptus species grow both phoracantha beetles are likely to be found CITATION Fox80 l 1033 (Fox, 1980); though attack is only centered towards dead, weakened and stressed trees and fallen limbs or cut logs CITATION Too35 l 1033 (Tooke, 1935). Furthermore, variations among Eucalyptus species in their likeliness to attack have been highly observed in several countries CITATION Cha69 l 1033 (Chararas, 1969) CITATION Dri75 l 1033 (Drinkwater, 1975.)CITATION POW78 l 1033 (Powell, 1978) CITATION Loy83 l 1033 (Loyttyniemi, 1983) CITATION Men84 l 1033 (Mendel, 1984.). In Portugal, it was evident in Mata do Escaropim, an arboretum area of 400ha situated near Lisbon in the south margin of the Tejo River. Here, 110 species of Eucalyptus, that were planted side by side in plots during the period 1953-1958 (reaching 1985), gave a picture of differential species colonization by P. semipunctata. CITATION Len88 l 1033 (Lencart, 1988) gave a report on Eucalyptus; bicostata, gigantea, globulus, ovata and viminalis that they were devastated by P. semipunctata larvae, on the other hand plots with other species, including E. tereticornis, showed no signs of colonization.

The function of olfactory responses by P. semipunctata and recurva to volatile components in host-finding behavior is well articulated CITATION Bar92 l 1033 (Barata, 1992.). Timber and leaves of Eucalyptus globulus are attractive to adult male and female P. semipunctata and recurva and wind tunnel studies tell us that odor-induced optomotor anemotaxis is involved in host finding behavior. In addition, survival of both P. semipunctata and recurva larvae varies significantly in logs of different Eucalyptus species and adult beetles are more highly attracted to the tree species that give the lowest mortality rate for their progeny CITATION Han93 l 1033 (Hanks, 1993).

Eucalypt species produce large amounts and numbers of volatile compounds, particularly isoprenoids (often called terpenes), that are found in glands abundantly distributed throughout the foliar parenchyma and bark CITATION Cha54 l 1033 (Chattaway, 1954.). The isoprenoid composition of Eucalyptus essential oils is to a large extent, under genetic control CITATION Dor91 l 1033 (Doran, 1991.). Variation among Eucalypt species in their content of such compounds has been related to host species preference by paropsine chrysomelid defoliators CITATION LiH93 l 1033 (Li, 1993) and has been useful in the chemotaxonomy of the genus CITATION LiH95 l 1033 (Li, 1995). Although, it is important to note that, in addition to the abundant essential oil isoprenoids, other volatile components would be expected in the air above growing trees.

3.0 METHODOLOGYPrimary and secondary data will be used in this study. Primary data will be collected from the experiment which will be conducted in the laboratory and field. And secondary data will be obtained from literature available on topics related to the study.

3.1 PROCEDUREExtraction and Analysis of volatile compounds from Eucalyptus species
The branches of different Eucalyptus species of about 1cm in thickness will be cut from commercial plantations and collected from each selected sample site in different locations. The sampling at either place shall be carried out during the dry season. The branches will be tagged and clearly labeled and carried to the laboratory. About 100g of bark will also be stripped off from selected stems and carried to the laboratory for analysis.

Distillation and extraction procedure
About 100 grams of different Eucalyptus species bark shall be placed in a 500ml round bottom flask and covered with 250 ml of distilled water heated to 60oC. This will be placed in a heated mantle and linked to one arm of the distillation and extraction apparatus; while a 50ml flask containing 20ml of methylene chloride, in a water bath at 40oC, will be connected to the other arm of the apparatus. About 10ml of methylene chloride will be added simultaneously into a tube located in the middle of the apparatus. Distillation shall be limited to 30 min. The condensing methylene chloride will be dried with 2g of anhydrous sodium sulphate for 15 min, and will be concentrated to a known volume. This will be used as crude extract. About 1 microliter of this concentrate will be injected into a Gas Chromatograph, coupled to a MS, for the identification and confirmation of volatile compounds. Once the volatile compounds are confirmed and analytical grade samples of the compounds identified (“standard chemicals”) will be used in the olfactometry.

Laboratory bioassay experiment
An olfactometer, made according to the design described by CITATION Pat93 l 1033 (Patterson, 1993) shall be used for the laboratory bioassay. During the bioassay a single Phoracantha female beetle shall be introduced to the center hole of the olfactometer. At the same time, a test volatile compound, or the crude extract, shall be incorporated into a small wick made out of cotton wool and pegged in the chamber of the olfactometer. The beetle’s response shall be measured by its attraction to the test chemical or the crude extract, after a given period of time. The olfactometric study shall be carried out in a dark chamber.

Field experiment
A Field experiment will be carried out at three different locations in commercial plantations of mature Eucalyptus trees between the ages 10 – 20 years. About three trees will be felled at each site to the ground as trap logs. The vertical sticky traps (cardboard sheets, 25 cm in diameter and 15 cm in height) shall be used in the field to trap the beetles. The sticky traps shall be set 10 meters apart and facing against the wind and placed at a height of about 1.5m above ground level. The beetle caught in each trap will be recorded. One side of the cardboard will be painted with red, blue, yellow or green color varnish paper to give a specific color for beetle attraction. The insect sticky glue will be applied to the surface of the colored side to form a sticky surface for trapping the beetles. The ordinary transparent sheets may be used as colorless traps.

3.2 STUDY AREAThe laboratory bioassay experiment will be conducted in the school laboratory right here at The Copperbelt University with coordinates;
(Latitude: – 12.8054° S, Longitude: – 28.2396° E).

The field experiment will be conducted at Chati plantation in Kalulushi with coordinates;
(Latitude: – 12° 49′ 59.99″ S, Longitude: -28° 04′ 60.00″ E).

3.3 SAMPLE SIZEThere will be three (3) treatments and 6 replications in the experiment. In the replications each treatment will have 6 larvae making a total of 36 larvae per treatment and the whole experiment will have a total of 108 experimental units.

3.4 TREATMENTSLaboratory bioassay experiment
1.Control: Crude extract incorporated into a small wick made out of cotton wool (Treatment 1)
2. An olfactometer with Eucalyptus leaf essential oils and a beetle introduced through the center hole (Treatment 2).

3.An olfactometer with Eucalyptus bark essential oils and a beetle introduced through the center hole (Treatment 3).

Field experiment
Trap logs with sticky colored card boards to attract beetles (Treatment 1)
Trap logs with sticky transparent card boards to attract beetles (Treatment 2)
EXPERIMENTAL DESIGNComplete randomized design (CRD) CITATION ASN l 1033 (A.S.Naik, 2013) will be used in order to avoid biasness and reduce systematic errors; the experiments will have 6 replications for each treatment.

T3 T6 T2
T5 T1 T4
T1 T4 T6
T6 T2 T3
T2 T3 T5
T4 T5 T1
3.6 DATA COLLECTIONResponse of beetles to Eucalyptus with respect to time
Total number of beetles that get trapped
Number of beetles that get caught on the colored card board
Number of beetles that get caught on the transparent card board
Number of beetles successfully reared in the laboratory
Number of beetle eggs collected from the field
3.6.1 DATA COLLECTION FORMName of experiment: e.g. control
Variable measured: e.g. diameter, height and time
Replication N#:e.g. 1
1 1
1 2
1 3
1 4
1 5
1 6
3.7 DATA HANDLING AND ANALYSISData on the olfactometric bioassay shall be analyzed using the Chi Square test. Analysis of variance shall be carried out on the responses of the beetles to the test compounds in the field. And the Analysis of Variance (ANOVA) will be performed using SPSS software CITATION Ark l 1033 (Arkkelin, 2014) for windows 10. Results will be expressed on graphs using excel for windows 10.
4.0 TIME FRAMEThe total time expected to be spent during the course of carrying out a research on this topic is a period of about 6 months, this is because of the nature of the experiment of trapping the Phoracantha beetles. The activities and the months in which they are expected to be done is summarized in the table below
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Arkkelin, D., 2014. Using SPSS to Understand Research and Data Analysis. s.l.:Valparaiso University, ValpoScholar.

Barata, E. N. a. A. J., 2000. Olfactory orientation responses of the eucalyptus woodborer, Phoracantha semipunctata, to host plant in a wind tunnel.. Physiol. Entomol. Submitted..

Barata, E. N. F. P. M. E. a. A. J., 1992.. Host-finding by Phoracantha semipunctata (Coleoptera: Cerambycidae): Host volatiles, electroantennogram recordings and baited field traps.. In: Proceedings of the 8th International Symposium on Insect-Plant Relationships.. Dordrecht: Kluwer Academic, pp. 133-135.

Bruce TJA, P. J., 2011. Perception of plant volatile blends by herbivorous insects finding the right mix. Phytochemistry , p. 72:1605–1611.

Chararas, C., 1969. Etude biologique de Phoracantha semipunctata E. (Coleoptere Cerambycidae xylophage) specifique des Eucalyptus en Tunisie et recherches sur la vitalite et I’ adaptation de ces essences.. Acad. Agric. Fr., pp. 47-57.

Chattaway, M. M., 1954.. The anatomy of bark. II.. Oil glands in Eucalyptus species.Aust. J. Bot., pp. 3:21-27..

Doran, J. C., 1991.. Commercial sources, uses, formation and biology. In: Eucalyptus Leaf Oils: Use, Chemistry, Distillation and Marketing.. Melbourne: Inkata Press, pp. 11-25.

Drinkwater, T. W., 1975.. The present pest status of eucalyptus borers Phoracantha spp. in South Africa.. Proc. I Congr. Entomol. Soc. S. Afr., pp. 119-129.

Duffy, E. A. J., 1963. A Monograph of the Immature Stages of Australasian Timber Beetles., London.: British Museum.

Fox, j. E. D. a. C. S. J., 1980. Notes on the tuart tree ( Eucalyptus gomphocephala) in the Perth area.. West. Aust. Nat., pp. 14:174-186.

Hanks, L. M. P. T. D. a. M. J. G., 1993. Host species preference and larval performance in the wood-boring beetle Phoracantha semipunctata F.. Oecologia., pp. 95:22-29..

Lencart, P., 1988. Algumas notas sobre a bio-ecologia e o controlo de Phoracantha semipunctata Fab. no Alto Alentejo. Project Report. , Portugal: Universidade de Tras-os-Montes e Alto Douro.

Li, H., 1993. Phytochemistry of Eucalyptus spp. and its role in insect-host-tree selection. PhD dissertation, Australia: Univesity of Tasmania.

Li, H. M. J. L. a. P. B. M., 1995. Variation in volatile leaf oils of the Tasmanian Eucalyptus species. 1. Subgenus Monacalyptus.. Biochem. Syst. Ecol, pp. 23:299-318.

Loyttyniemi, K., 1983. Flight pattern and voltinism of Phoracantha semipunctata (Coleoptera, Cerambycidae) in a semihumid tropical climate in zambia.. Ann. Entomol. Fenn., pp. 49:49-53..

Mendel, Z. G. Y. a. M. Z., 1984.. Studies on the phenology and some mortality factors of the eucalyptus borer Phoracantha semipunctata in Israel.. La-Yaaran., pp. 34:41-43..

Osuolale Peter Popoola, K. K. A. T. M. O. A. W. A., 2015. A Quadratic Regression Analysis of the Effect of Three Levels of NPK Fertilizer on the Yield of the Yellow Maize. American Journal of Computational Mathematics..

Paine, T. D. J. G. M. E. O. P. a. L. M. H., 2001. Inßuence of host log age and refuge from natural enemieson colonization and survival of Phoracantha semipunctata.. Entomol. Exp. Appl., pp. 98: 157-163.

Patterson, M. Q. S. J. C. C. W. S. e. a., 1993. Detection thresholds for an olfactory mixture and its three constituent compounds. Chemical Senses, 18(6).

Powell, W., 1978. Colonization of twelve species of Eucalyptus by Phoracantha semipunctata (F.) (Coleoptera: Cerambycidae) in Malawi.. Bull. Entomol. Res., pp. 68:621-626.

Tooke, F. G. C., 1935. The Phoracantha beetle.. Bull. Dept. Agric. For. S. Afr., pp. 142:33-39.