Thesis Template

TAXONOMY, DISTRIBUTION AND PEST

STATUS OF PLUTELLA SPECIES

AUSTRALIA AND NEW ZEALAND

Kariyawasam Haputhanthri Kankanamge Tharanga Niroshini

Submitted in fulfilment of the requirements for the degree of

Master of Applied Sciences (Research)

School of Earth Environmental and Biological Sciences Science and Engineering Faculty Queensland University of Technology 2018

Keywords

Adults, ANOVA, Bayesian analysis, CO1 barcode gene, crops, DNA, genitalia morphological features, host plant, larvae, light trap, maximum likelihood, measurements, PCR, phylogenetic analyses, Plutella australiana, Plutella xylostella, R statistical analyses, Sanger sequencing, taxonomy.

Abstract

The diamondback moth (DBM), Plutella xylostella L. (Lepidoptera: Plutellidae), is the most destructive agricultural pest in the word causing damage to brassica crops such as cabbage, kale, broccoli, and cauliflower. Its global distribution, movement over long distances and rapid evolution of insecticide resistance make this a key pest of international importance. P. xylostella was introduced to Australia in 1882 and has become widely distributed in Australia.

Despite reports of low levels of genetic variation in the Australian population (Endersby et al., 2006), more recent molecular studies indicated the presence of variants within the Australian Plutella population. A study of allozymes in P. xylostella populations from 14 locations worldwide included specimens from 5 different locations in Australia (Adelaide, Brisbane, North Queensland, Melbourne and Sydney), and found significant differences within the samples from Australia (Pichon et al., 2006). Similarly, Roux et al. (2007) using the inter simple sequence repeat (ISSR) marker showed a genetic differentiation between Melbourne and Sydney P. xylostella populations.

In 2013, a new taxon, Plutella australiana, was described (Landry & Hebert, 2013) based on 8.6% sequence divergence in the ‘barcode’ region of the mitochondrial cytochrome c oxidase 1 gene (CO1) and differences in the morphology of the genitalia in both males and females. The new taxon was identified as broadly distributed in southern and eastern Australia. However, there were no larval collections, leaving the host plants and possible pest status unknown. In addition, the description of the new and potentially endemic taxon created difficulties in the import and release of biological control applications, and potential difficulties in pest management practices and market access.

This study identified and addressed gaps in knowledge, with the main aim being to clarify the differentiation of P. australiana from P. xylostella and to increase knowledge of its distribution and host plants. CO1 barcode sequence data were used in the identification of both taxa, including phylogenetic analyses (Maximum ii

likelihood and Bayesian inference). Morphological features of female and male genitalia were measured to determine statistical variance and examined to identify reliable diagnostic features. Distribution of P. australiana at the regional scale was evaluated from field collections, including individuals from New Zealand. Collections of larvae from both crops and wild brassicas were conducted to identify host plants and contribute to knowledge of the possible pest status of P. australiana. These examinations contribute to the better understanding of possible risks to Australian brassica production, and to inform potential pest management strategies.

The results showed that P. xylostella and P. australiana are two distinct taxa based on CO1 data. Examination of key morphological features showed that only two features, the curvature of the tubular projection in P. australiana and the presence of raised folds surrounding the antrum in P. xylostella, and both only in females, are reliable as diagnostic tools. Although some measured features are statistically significantly different overall between the two populations, the overlap in the variance indicates that those features cannot be used as diagnostic tools. Similarly, other characteristic features proposed by Landry and Hebert (example: sinuation in the ventral margin of the valva) were found to be not reliable for identification of the two taxa.

Light trap collections of adults show that the two taxa are sympatric in most locations, including Tasmania. However, larvae of P. australiana were present in only two collections: on cabbage (Brassica oleracea) in Theresa Park, NSW in 2015 and on field mustard weeds (Brassica rapa) amongst a kale crop in Werombi NSW in 2015. This study is the first to describe the occurrence of P. australiana larvae on cabbage and field mustard. The preference of P. australiana for weedy B. rapa over kale in one site suggests that emergence of P. australiana as a pest of canola requires investigation.

The outcomes of this thesis have been to broaden the knowledge of the new taxon, P. australiana including morphological features that can be used in order to differentiate P. xylostella and P. australiana. The results contribute to the better understanding of possible risks of the new taxon to Australian brassica production, and to inform potential pest management strategies.

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Table of Contents

Keywords ...... i

Abstract ...... ii

Table of Contents ...... v

List of Figures ...... viii

List of Tables ...... xiii

List of Abbreviations ...... xvii

Statement of Original Authorship ...... xviii

Acknowledgements ...... xix

Chapter 1: General Introduction and Literature Review ...... 1

1.1 Background ...... 1

1.1.1 Research problem and aims ...... 2

1.1.2 Thesis outline ...... 3

1.2 Literature Review ...... 4

1.2.1 Life history ...... 4

1.2.2 Distribution ...... 8

1.2.3 Host plants ...... 10

1.2.4 Pest management ...... 14

1.2.5 Taxonomy ...... 17

1.2.6 A new Plutella taxon in Australia...... 19

Chapter 2: Molecular and morphological examination of Plutella species in

Australia and New Zealand ...... 25

2.1 Introduction ...... 25

2.2 Materials and Methods ...... 28

2.2.1 Sampling ...... 28

2.2.2 Molecular analysis ...... 31

2.2.3 Morphology ...... 33

2.3 Results ...... 39

2.3.1 CO1 sequence data...... 39

2.3.2 Morphology ...... 43

2.4 Discussion ...... 49

Chapter 3: Host plants and distribution of Plutella species in Australia ...... 53

3.1 Introduction ...... 53

3.2 Materials and Methods ...... 56

3.2.1 Sampling ...... 56

3.2.2 Molecular Analysis ...... 56

3.2.3 Morphology ...... 57

3.3 Results ...... 59

3.3.1 CO1 sequence Data ...... 59

3.3.2 Host plants of P. xylostella and P. australiana ...... 59

3.3.3 Morphology ...... 62

3.4 Discussion ...... 76

Chapter 4: General Discussion ...... 79

4.1 CO1 ‘barcode’ analysis of Australian and New Zealand Plutella taxa ...... 80

4.2 Diagnostic morphological features ...... 82

4.3 Summary and conclusion on the taxonomy of P. xylostella and P. australiana ...... 84

4.4 Distribution, host range and pest status of the two taxa ...... 85

4.4.1 Distribution ...... 85

4.4.2 Host preference and pest status of P. australiana...... 86

4.4.3 Summary and conclusions on the distribution, host range and pest status of

the two taxa...... 87

4.5 Pest management ...... 88

4.6 Limitations and recommendations ...... 88

References ...... 91

Appendices ...... 104

Appendix A ...... 104

Appendix B ...... 105

Appendix C ...... 109

Appendix D ...... 113

Appendix E ...... 114

Appendix F...... 115

Appendix G ...... 116

Appendix H ...... 118

Supplementary Materials ...... 121

vii

List of Figures

Figure 1.1 Diamondback moth eggs...... 5

Figure 1.2 Fourth instar larvae of diamondback moth...... 5

Figure 1.3 Swede crops in a community garden damaged by DBM larvae...... 6

Figure 1.4 Diamondback moth pupa on a red cabbage leaf...... 6

Figure 1.5 A) Lateral view of a DBM adult and B) dorsal view of a DBM

showing the diamond pattern of a DBM adult...... 7

Figure 1.6 World distribution of P. xylostella, the diamondback moth. Blue =

widespread, Black = present. Although some countries are not marked,

DBM may be present (CABI, 2016)...... 9

Figure 1.7 Major biological control agents of P. xylostella (Sarfraz et al., 2005).

...... 15

Figure 1.8 Sites in Australia where specimens of P. xylostella (red) and P.

australiana (blue) have been collected. The circles show the proportion

of the two species at each site. These records only include specimens

identified through DNA barcode analysis (Landry & Hebert, 2013)...... 20

Figure 1.9 Picture from Dugdale (1973) showing the curved tubular projection

(12) in a female genitalia from a diamondback moth specimen collected

from New Zealand...... 22

Figure 2.1 Distribution of diamondback moth in Australia. Blue = widespread,

black = present (CABI, 2016)...... 25

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Figure 2.2 Neighbor joining tree (nodes collapsed) based on Kimura-2-

parameter distances for the barcode region of the cytochrome c oxidase

1 gene. Specimens are labelled by the Australian state or by country of

origin and bracketed numerals indicate the number of specimens from

each site (taken from Landry and Hebert (2013))...... 26

Figure 2.3 A) Ranger moth light trap in a cabbage field and B) Insects trapped

in the light trap. Moths of Plutella spp. are circled in yellow...... 29

Figure 2.4 Measurements of both female and male adult genitalia characteristics

recorded to determine the statistical variance across taxa (Scale bars =

200 μm). Images were taken by Tharanga Kariyawasam...... 37

Figure 2.5 Distribution of DBM adults caught in light traps at Samford (QLD),

Hobart (TAS), Theresa Park (NSW), Werombi (NSW), Mowbray Park

(NSW), Birkdale (QLD), Gatton (QLD) and Laidley (QLD). Numbers

within bars represent the individuals identified and assigned to relevant

taxa (see Table 2.1. for total number of individuals)...... 40

Figure 2.6 Bipartition maximum likelihood (ML) tree with bootstrap values.

The tree was collapsed to remove low supported nodes (≥75%) and the

nodes were further collapsed (shape of the clade) because of the large

number of specimens assigned to each taxon. See Appendix D for the

original phylogenetic tree...... 41

Figure 2.7 Bayesian analysis with posterior probabilities. The tree was collapsed

to remove low supported nodes (≥75%) and the nodes were further

collapsed (shape of the clade) because of the large number of specimens

assigned to each taxon. See Appendix E for the original phylogenetic

tree...... 42

Figure 2.8 Density plots showing the distribution of measurements taken for a)

tubular projection length (TPL) and b) sternite 7 length (S7L)

parameters of females showing an overlap between Australian P.

australiana (AA), Australian P. xylostella (AX) and New Zealand P.

xylostella (NX)...... 46

Figure 2.9 Density plots showing the distribution of measurements taken for (a)

phallus length (PL) and (b) vinculum saccus length (VSL) parameters

of males showing an overlap between Australian P. australiana (AA),

Australian P. xylostella (AX) and New Zealand P. xylostella (NX)...... 48

Figure 3.1 DBM larvae damaging A) cabbage leaf, B) head formation of red

cabbage...... 54

Figure 3.2 Distribution of larvae from field collections in Hobart (TAS), Theresa

Park (NSW), Werombi (NSW), Gatton (QLD), Laidley (QLD),

Currumbin (QLD) and Birkdale (QLD). Numbers within bars represent

the individuals identified and assigned to relevant taxa (for total

numbers see Table 3.1.)...... 60

Figure 3.3 Field mustard (B. rapa) (above) and the cabbage field (below) that

P. australiana larvae were collected. In order from left to right are the

field mustard plant, its flower, larvae feeding on the leaf, pupae found

on the stem and the cabbage field where P. australiana larvae were

collected...... 61

Figure 3.4 Density plots showing the distribution of measurements taken for a)

whole length (WL), b) whole width (WW), c) tubular projection length

(TPL) and d) sternite 7 length (S7L) parameters of females showing an

overlap between P. xylostella and P. australiana. However, mean

values are significantly different...... 65

Figure 3.5 Density plot showing the distribution of measurements taken for

phallus length (PL) parameter of males showing an overlap between P.

xylostella and P. australiana. However, mean values are significantly

different...... 67

Figure 3.6 Density plots showing the distribution of measurements taken for a)

whole length (WL), b) upper part length (UPL) and c) sternite 7 length

(S7L) parameters showing an overlap between Australian P. xylostella

adults (AUS adults), New Zealand P. xylostella adults (NZ adults) and

P. xylostella larvae. However, mean values are significantly different...... 69

Figure 3.7 Density plots showing the distribution of measurements taken for

whole width (WW) and tubular projection length (TPL) parameters of

females, showing an overlap between Australian P. australiana adults

from light traps and P. australiana adults reared from larvae. However,

mean values are significantly different...... 71

Figure 3.8 Density plot showing the distribution of measurements taken for

phallus length (PL) parameter of males, showing an overlap between P.

xylostella adults from light traps and P. xylostella adults reared from

larvae...... 73

Figure 3.9 Density plots showing the distribution of measurements taken for

whole length (WL), valva length (VL) and phallus length (PL)

parameters of males, showing an overlap between P. australiana adults

from light traps and P. australiana adults reared from larvae. However,

mean values are significantly different...... 75

xii

List of Tables

Table 1.1 Worldwide distribution of Plutella species. (Information taken from

(Robinson & Sattler, 2001)...... 9

Table 1.2 Known host plants of P. xylostella (Information taken from Sarfraz et

al. (2006))...... 12

Table 2.1 Locations of DBM adult collections and number of individuals taken

for genetic and morphological analyses. Specimens not taken for

morphological analyses are marked as ‘/’ and unknown GPS

coordinates are marked as ‘-‘...... 30

Table 2.2 Morphological features of DBM female genitalia examined in this

study to identify diagnostic features. Features are circled in red. Images

were taken by Tharanga Kariyawasam...... 35

Table 2.3 Morphological features of DBM male genitalia examined in this study

to identify diagnostic features. Features are circled in red. The ventral

view of P. australiana and lateral views of the valva were taken from

Landry and Hebert (2013). The remaining images were taken by

Tharanga Kariyawasam...... 36

Table 2.4 Images show the appearance of the vinculum saccus in two males

where one had the characteristic morphology of P. australiana but were

identified as P. xylostella from CO1 sequence data and the other had

the characteristic morphology of P. xylostella but were identified as P.

australiana from CO1 sequence data. These specimens were collected

from Hobart and Theresa Park respectively...... 44

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Table 2.5 Mean values ± SD of parameters for each female population are shown

in micrometers (μm). P-value of the one-way ANOVA (at 0.95

confidence intervals) are presented which showed a significance

difference for the TPL (tubular projection length) parameter and the

S7L (sternite 7 length) parameter. Statistically significant codes: ***P

< 0.001, **P < 0.01...... 45

Table 2.6 Mean values ± SD of parameters for each male population are shown

in micrometers (μm). P-value of the one-way ANOVA (at 0.95

confidence interval) is presented which showed a significance

difference for the PL (phallus length) parameter and VSL (vinculum

saccus length) parameter. Statistically significant codes: *P < 0.05,

***P < 0.001...... 47

Table 3.1 Larvae collection details including location, crop type and number of

individuals taken for genetic and morphological analyses. Specimens

not taken for morphological analyses are marked as ‘/’ and unknown

GPS coordinates are marked as ‘-‘...... 57

Table 3.2 The image shows the appearance of the vinculum saccus of a male

that had the characteristic morphology of P. australiana but was

identified as P. xylostella from CO1 sequence data. The larva was

collected from B. rapa weeds in Werombi NSW in 2015...... 63

Table 3.3 Mean ± SD values of parameters for each female population are shown

in micrometres (μm). P-value of the one-way ANOVA (at 0.95

confidence intervals) is presented which showed a significance

difference for the whole length (WL), whole width (WW) and tubular

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projection length (TPL) parameters. Statistically significant codes: *P

< 0.05, **P < 0.01...... 64

Table 3.4 Mean ± SD values of parameters for each male population are shown

in micrometres (μm). P-value of the one-way ANOVA (at 0.95

confidence intervals) is presented which showed a significance

difference for the phallus length (PL) parameter. Statistically

significant codes: ***P < 0.001...... 66

Table 3.5 Mean ± SD values of parameters for each female population are shown

in micrometres (μm). One-way ANOVA (at 0.95 confidence intervals)

is presented which showed a significance difference for the whole

length (WL) and upper part length (UPL) parameters. Statistically

significant codes: *P < 0.05...... 68

Table 3.6 Mean ± SD values of parameters for each female P. australiana

population are shown in micrometres (μm). One-way ANOVA (at 0.95

confidence intervals) and Kruskal Wallis test are presented which

showed a significance difference for the whole width (WW) and tubular

projection length (TPL) parameters. Statistically significant codes: *P

< 0.05...... 70

Table 3.7 Mean ± SD values of parameters for each male P. xylostella

population are shown in micrometres (μm). One-way ANOVA (at 0.95

confidence intervals) is presented which showed a slight difference for

the phallus length (PL) parameter. Statistically significant codes: ‘.’0.1.

...... 72

Table 3.8 Mean ± SD values of parameters for each male P. australiana

population are shown in micrometres (μm). One-way ANOVA (at 0.95

confidence intervals) is presented which showed a significance

difference for the whole length (WL), valva length (VL) and phallus

length (PL) parameters. Statistically significant codes: *P < 0.05, **P

< 0.01,***P < 0.001...... 74

xvi

List of Abbreviations

ANOVA analysis of variance BLAST basic local alignment search tool bp base pairs DBM diamondback moth DDT dichlorodiphenyltrichloroethane DNA deoxyribonucleic acid MEGA molecular evolutionary genetics analysis MUSCLE multiple sequence comparison by log-expectation PCR polymerase chain reaction RAxML randomized axelerated maximum likelihood SD standard deviation μm micrometre

xvii Statement of Original Authorship

The work contained in this thesis has not been previously submitted to meet requirements for an award at this or any other higher education institution. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made.

Signature: QUT Verified Signature

Date: February 2018

xviii

Acknowledgements

I am thankful to all the people who have encouraged, helped and contributed along the way to accomplish my goals and completing this thesis.

My biggest thanks is to my husband for the immense encouragement, love and dedication given during my study - starting from moving to Australia with me; to staying by my side in all the hardest moments of my candidature including health conditions I had to resolve along the way; helping in my travelling and field collections; not complaining when I worked in the lab till late at night and during the weekends. You are my strength and I also appreciate your patience during the time of my thesis writing.

My sincere and heartfelt thank you goes to my parents with whom my achievements would have not been possible without them by my side and their unconditional love, immense support, encouragement and believing in me. Love to my sister who has always been my biggest supporter especially throughout the time of this study and thanks to my brother-in-law for his kind support.

Many thanks go to my principal supervisor associate professor Caroline Hauxwell for her immense guidance, time, contribution, editing and advice given to me to undergo this study and towards the completion of my study and thesis writing. Under her guidance I have learned a lot about arranging, planning and accomplishing tasks and especially in conducting field work and going to new places in Australia, especially at times when driving by myself along the way. I discovered many beautiful places in Australia as an international student.

A big thanks to my associate supervisor Dr Susan Fuller for her immense help, encouragement and dedication of her time in editing my thesis. A sincere thank you for my external supervisor, Associate Professor Stephen Cameron for his guidance and help, initially as my associate supervisor till he moved to the US. Thanks to my former supervisor Dr Mark Schutze for his help and advice.

xix

I also thank Professor Acram Taji for her timely and immense guidance given from the beginning of my candidature whenever I sought her advice. My gratitude to Dr Tanya Scharaschkin for her help in answering the phylogenetic problems I had as a new researcher in that area and also her help in the identification of plant materials I collected during my field collections. My sincere thanks to Dr Mattew Krotch for his help and guidance given to me whenever I needed to understand or solve issues I had in my study.

It was a great experience in meeting and connecting to many people in order to find fields to conduct light trapping and collect larvae and also to gather information needed and samples to support my study. I was given support from many lovely people who arranged, and assisted me in field collections in Sydney, Tasmania and Brisbane. For that thank you to Andy Ryland, Jason Lynch, Lionel Hill, Catherine Byrne, Lara Senior, Gavin Berry and Achinda. For allowing me to conduct the field collections in their farms thank you to Matt, Eddie, Franco, David, Wade and Mulgowie farming company. For providing me with valuable information, document arrangements and sending samples from New Zealand I extend my appreciation to John Dugdale, Graham Walker, Philippa Stevens and others who helped in the collections and other arrangements. For providing information I thank Ted Edwards and Christian Mille. I wish to appreciate the help given by Desley Tree in showing me how to conduct slide preparations.

I am thankful to the QUT EEBS laboratory and technical staff for their immense support in providing necessary equipment and support during my laboratory and field work. For that thank you to Amy Carmichael, Anne-Marie McKinnon, Karina Pyle, Mark Crase, and Vincent Chand. My gratitude to QUT members; EEBS research student support team finance officers and research student centre for the help given to me during my candidature. My extended appreciation to Karyn, Sophie and Christian from the QUT Academic Language and Learning Services for their guidance and kind support in reading and correcting drafts of my thesis. My research group members and peers in the writing circle sessions helped me develop my writing skills. Thank you to the QUT International Student Services and disability center for providing me the support and guidance I needed at the time. I also thank QUT EEBS school and faculty members for any help given to me during my candidature. xx

Thank you for all the members in the invertebrate microbiology group especially Robert and Andrew for assisting me in the field work, molecular and DBM colony work. Special thanks to Melodina Fabillo for her great help, guidance and patience in teaching and showing me phylogenetic analyses methods. Similarly, I thank Purnika, Joshua and Thita for their great help to tackle the problems in R statistics and interpretation of the results. Thanks for the members in the fruit fly research group who have helped me in various ways during my study. Friends whom I have exchanged life experiences and research experiences were valuable in keeping me going through my study. For that thanks to Purnika, Thita, Lixin, Rak, Melody, Sarah, Noor, Aisha, Naimul, Joshua, Savindi, Sasha, Hernan, Christi, Karma and other friends outside QUT.

Finally, I express my gratitude to the Lion Center for providing the living stipend support during my study and QUT for the opportunity to pursue my studies and for providing me the tuition fee waiver.

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Chapter 1: General Introduction and Literature Review

1.1 Background

The Lepidoptera (butterflies and moths) are one of the largest and most widespread insect orders, yet most species are awaiting formal description (Zhang, 2011). The superfamily Yponomeutoidae (clade Ditrysia) contain around 1800 species worldwide and include notable pest species, including the leek moth (Acrolepiopsis assectella: Glyphipterigidae), small ermine moths (Yponomeuta spp.) and the diamondback moth (Plutella xylostella L. Plutellidae) (Heppner, 1998; Zhang, 2011). Around 58 species are known in the genus Plutella (Encyclopedia-of-Life; Robinson & Sattler, 2001).

The diamondback moth (DBM), P xylostella is the most destructive insect pest of Brassica crops such as cabbage, kale, broccoli, and cauliflower, with global damage and control costs estimated to be US$ 4-5 billion annually (Furlong et al., 2013). Diamondback moths have developed resistance to most insecticides (Atumurirava et al., 2011; Sun et al., 1986; Zhou et al., 2011) and were reported as the first species to develop resistance to some toxins of Bacillus thuringiensis (Tabashnik et al., 1987; Talekar & Shelton, 1993). Natural enemies including parasitoids, arthropod predators, viruses, microsporidia, pathogenic fungi and bacteria have been explored as biological controls (Furlong et al., 2013). Classical biological control introduces and establishes natural enemies where exotic pests have been introduced but without their natural enemies (Waterhouse & Sands, 2001). These biological controls could potentially cause damage to native species, and introduction of biological controls into Australia requires intensive and extensive non-target testing before release under the Biosecurity act (2015).

Since first reported in Australia (Tryon, 1889), P. xylostella has become common and widely distributed, and is resistant to synthetic pyrethroid insecticides in all Australian states (AgricultureVictoria, 1996; Endersby et al., 2011). The release of biological

controls against the exotic P. xylostella in Australia has been more challenging with the description of a new, closely related taxon, Plutella australiana, by Landry and Hebert (2013), proposed this as a morphologically-cryptic Australian native taxon. This proposed new taxon was described based on 8.6% sequence divergence in the barcode region of the mitochondrial cytochrome oxidase 1 (CO1) gene and the differences in the morphology of genitalia in both females and males.

The distribution of the new taxon was described as southern and eastern Australia but was not found in Tasmania. Further sampling was required to confirm the presence or absence of P. australiana in other parts of Australia. The description was based only on adults from light traps, with no larval collections, and so host plant use and possible pest status of the new taxon were unknown. The only subsequent host record for P. australiana was on Lincoln weed from South Australia in 2015 (Perry et al., 2015). Consequently, the species status, host plants, pest status and wider distribution of the newly described P. australiana remains largely unknown, and this has created uncertainty for future biological control applications, pest management practices and market access.

1.1.1 Research problem and aims

The main aim of this thesis is to clarify the differentiation of P. australiana from P. xylostella and to increase knowledge of its distribution and host plants. CO1 barcode sequence data were used in the identification of both taxa, including phylogenetic analyses (Maximum likelihood and Bayesian inference). Morphological features of female and male genitalia were examined and measured to identify reliable diagnostic features. Distribution of P. australiana at the regional scale was evaluated from field collections, including individuals from New Zealand. Collections of larvae from both crops and wild brassicas were conducted to identify host plants and contribute to knowledge of the possible pest status of P. australiana. These examinations contribute to the better understanding of possible risks to Australian brassica production, and to inform potential pest management strategies.

The following specific research aims were identified for this study;

1) To clarify the identity of the two taxa based on CO1 barcode, with an increased sample size from the original published study. 2) To identify which morphological features can be used to differentiate the two taxa by conducting a detailed morphological comparison of the adult genitalia. 3) To add to the current knowledge of distribution by examination of samples from the East Coast of Australia, Tasmania and New Zealand. 4) To collect larvae to identify the host plants of P. australiana and contribute to understanding of the possible pest status of the new taxon. 5) To consider the implications for management of Plutella spp. in Australia and to inform management strategies.

The outcomes of this thesis have been to broaden the knowledge of the new taxon, P. australiana including its potential threat to Australian brassica production, and to identify morphological features that can be used in order to differentiate P. xylostella and P. australiana. The results contribute to the better understanding of possible risks of the new taxon to Australian brassica production, and to inform potential pest management strategies.

1.1.2 Thesis outline

This thesis has four chapters; Chapter 1 (this chapter): Contains the background to the thesis, research questions and aims, the thesis outline and a literature review.

Chapter 2: This chapter addresses the first three aims of this study. It includes molecular (CO1 barcode and phylogeny), identification and measurement of key features of the genitalia of both Plutella taxa in Australia and New Zealand specimens. This chapter focusses on the description of adult specimens collected from light traps.

Chapter 3: This chapter addresses the fourth and fifth aims of this study. It focuses on the field collected larvae from brassica crops and weeds from different locations in

Australia including Tasmania and New Zealand. It includes molecular (CO1 barcode) and measurement of morphological features of adults reared from larvae collected in the field with a comparison with the adult individuals included in chapter 2. Identification of P. australiana host plants, pest status of the new taxon and its implication for pest management are included.

Chapter 4: Provides the general discussion, combining the results obtained in chapter 2 and 3 of this study. Limitations and recommendation for future works are included.

1.2 Literature Review

This literature review details the life history, distribution, host plants, pest management and taxonomy with a special reference to the Australian Plutella taxa. This review identifies the gaps which need to be addressed leading to the main research problem of the current study.

1.2.1 Life history

The life cycle of Plutella xylostella, the diamondback moth (DBM) ranges from 14 to 50 days depending on temperature (Waterhouse & Sands, 2001). Adults emerge in early summer, laying eggs on all parts of the brassica plant but mainly on the upper surface of the leaves. After hatching, there are four larval instars followed by pupation in open cocoons. Many overlapping generations can be found in the course of a year during a single brassica vegetable or canola crop cultivation, with all life stages of the DBM are present in the crop at the same time, and they complete multiple generations each year (Furlong et al., 2008; Talekar & Shelton, 1993).

Eggs

Figure 1.1 Diamondback moth eggs.

The eggs are pale yellow, approximately 0.5mm in length and laid either singly or as clusters on all parts of the plant (Figure 1.1). The eggs hatch in 4 to 8 days depending on temperature (D. Harcourt, 1957).

Larvae

Figure 1.2 Fourth instar larvae of diamondback moth.

First instar larvae are approximately 1-2mm in length whereas the fourth instars (Figure 1.2) are approximately 12mm in length and pale green in colour. The head capsule is pale brown and the body, green and segmented. Whenever the host plant is

disturbed the larvae retreat backwards and drop down from the host plant using a silky tread.

Although the first instar larvae are leaf miners and leave pale, tunnel-like patches on the leaves, later larval stages are surface feeders chewing out small irregular holes. The larva completes development in 10 to 30 days depending on the temperature and it is this time that is the problematic stage in terms of causing damage to the Brassica crops (Figure 1.3).

Figure 1.3 Swede crops in a community garden damaged by DBM larvae.

Pupae

Figure 1.4 Diamondback moth pupa on a red cabbage leaf.

Pupae can be seen in white silky cocoons generally fastened to the plant parts on the host plant (Figure 1.4) (CABI, 2016). Pupae are approximately 10mm in length and turn green to brown before adult emergence. Emerging will take 4 to 10 days depending on the temperature. If the cocoon is damaged or removed the pupae survival is very low.

Adults

Figure 1.5 A) Lateral view of a DBM adult and B) dorsal view of a DBM showing the diamond pattern of a DBM adult.

Adults are 10-12 mm in size and greyish brown in colour with a distinct beige band pattern along the inner side of their forewings which when viewed dorsally, appear as

three or four diamond shaped areas when at rest, giving the moth its common name (Figure 1.5 A, B). The diamond shapes are more distinct in males than those of females.

Adults become active just before dusk and stay inactive during the day unless disturbed. Mating begins at dusk on the day of emergence (CABI, 2016). Almost 95% of females begin laying eggs on the day of emergence (CABI, 2016); a process that lasts 10 days with the number of eggs laid per female ranging from 159 (D. Harcourt, 1957) to 288 (Ooi & Kelderman, 1979). Sivapragasam and Heong (1984) reported that temperature has a significant effect on adult survival, oviposition rates and generation (CABI, 2016). Adults do not fly long distances within a crop, they only fly only around 13 m - 35 m within a crop field (Mo et al., 2003). They migrate long distances carried by the winds for about 1500 km at 400 km - 500 km per night (Chapman et al., 2002).

1.2.2 Distribution

Global distribution

The origin of P. xylostella (syn. P. maculipennis) is not clear. It may have originated in the Mediterranean (Hardy, 1938), Africa (Kfir, 1998) or Asia (Liu et al., 2000), based on the number of endemic brassicas, molecular data and the diversity of parasitoid species identified from these regions. A recent study favoured an African or possibly Asian origin over a European origin based on high haplotype diversity, particularly in the African population, and mismatch analysis supported a more recent spread into North America, Australia and New Zealand (Juric et al., 2017).

Plutella xylostella now has a cosmopolitan distribution (Figure 1.6, Table 1.1) and can be found wherever suitable host plants are found (Shelton, 2001). P. xylostella is known to move over long distances in air currents (CABI, 2016), with distances of 1500 km at 400 km - 500 km per night (Chapman et al., 2002). Moreover, the movement of insecticide resistant individuals between countries can have serious implications for its pest control (CABI, 2016).

Figure 1.6 World distribution of P. xylostella, the diamondback moth. Blue = widespread, Black = present. Although some countries are not marked, DBM may be present (CABI, 2016).

Table 1.1 Worldwide distribution of Plutella species. (Information taken from (Robinson & Sattler, 2001). Number Plutella species Countries of presence 1 P. xylostella (P. meculipennis) Cosmopolitan (except extreme alpine areas and Antarctic region) 2,3 P. antiphona, P. psammochroa New Zealand 4 , 5, 6 P. notabilis, P. omissa , P. armoraciae USA (Washington) 7 P. geniatella Switzerland 8, 9 P. polaris, P. haasi Norway 10 P. mariae Russia 11, 12, 13 P. capparidis, P. noholio, P. kahakaha Hawaii 14 P. porectella Europe, North America and South America 15 P. balanopis South Africa 16, 17 P. deltodoma, P. diluta Chile 18 P. canaella Italy 19, 20 P. acrodelta, P. nephelaegis Argentina 21 P. rectivittella Colombia 22 P. formicatella Seychelles

Australian distribution

Plutella xylostella was first reported in Australia in1882 in Queensland (Tryon, 1889) and is now common and widely distributed within Australia (French, 1893; Lea, 1895; Nielsen et al., 1996; Thompson & Moore, 1895). The introduction and distribution of P. xylostella in Australia was thought to occur from the imported cabbage via passenger steamers (Tryon, 1889). Plutella xylostella is now a significant economic pest of brassicas, reported to attack up to 136,000 hectares of brassica vegetable crops (Shelton, 2001), and in canola (cultivars of Brassica rapa L. and B. napus L.), which is widely grown in South-east Australia and Western Australia. Australia is the world’s second largest exporter of canola (RIRDC, 2005-06) and canola is the third largest winter crop in Australia, thus management of P. xylostella is of considerable importance. Moreover, resistance to synthetic pyrethroid insecticides has been detected in populations of DBM in all Australian states (AgricultureVictoria, 1996; Endersby et al., 2011). This rapid and widespread evolution of resistance supports the need for an effective integrated resistance management strategy, as well as an integrated pest management strategy.

1.2.3 Host plants

Plutella xylostella is known as one of the most destructive insect pests of Brassicaceous crops worldwide (Ahuja et al., 2010; Furlong et al., 2013). The Brassicaceae family contains 380 genera and over 3000 species of cultivated and wild plants (Heywood, 1993; Warwick et al., 2003). This family contains economically important crops such as cole crops (cabbage, cauliflower), oilseeds (canola, mustard) and root vegetables (radish, turnip) (Muhammad et al., 2005).

Wild cruciferous plants act as a bridging host for P. xylostella between crops, and a few studies have examined the presence of P. xylostella on wild cruciferous and non- cruciferous host plants (Barker et al., 2001; Begum et al., 1996; Furlong et al., 2013; Löhr & Rossbach, 2001; Robinson & Sattler, 2001; Sarfraz et al., 2006; Sarfraz et al., 2011) (Table 1.2). Wild plants affect the developmental and reproductive parameters in P. xylostella (Begum et al., 1996; Harcourt, 1986; Muhamad et al., 1994; Sarfraz, Dosdall, & Keddie, 2010; Shelton & Nault, 2004; Talekar & Shelton, 1993; Yamada,

1983), and P. xylostella grown on wild crucifers were found to fly for longer (Begum et al., 1996). For these reasons, (Sarfraz et al., 2011) suggested that monitoring and controlling P. xylostella in weed species even before crops are cultivated may help controlling this pest in the fields.

Brassicas contain glucosinolates and sulphur-containing secondary plant compounds which stimulate DBM feeding and oviposition (Furlong et al., 2013; Marazzi et al., 2004). Similarly, plant volatiles, waxes, host plant nitrogen content, leaf morphology and leaf colour, or a combination of these factors, have been reported to stimulate the oviposition and feeding of P. xylostella (Sarfraz, Dosdall, & Keddie, 2010; Sarfraz et al., 2006; Stoner, 1990). Moreover, soil fertility levels are known to affect the oviposition and herbivory in P. xylostella (Sarfraz et al., 2006) similar to studies showing that the level of nutrients in the leaves affect parameters such as development and survival in other insects such as aphids and leaf mining flies (Björkman, 2000; Bruyn et al., 2002).

Plants such as B. rapa and B. napus were found to be preferred host plants among P. xylostella from early years until now (Brown et al., 1999; Clarke, 1971; Endersby et al., 2004; Perry et al., 2015; Talekar & Shelton, 1993). Those host plants including cabbage are known to contain green leaf volatiles, isothiocyanates, nitriles, dimethyl trisulfide, and terpenes which may be the reason to be more attractive to P. xylostella (Girling et al., 2011; Kugimiya et al., 2010).

Table 1.2 Known host plants of P. xylostella (Information taken from Sarfraz et al. (2006)).

Species/cultivar Common name(s) Selected reference(s) (i) Cultivated cruciferous host plants of P. xylostella Idris and Grafius (1996), and Brown et al. Brassica napus L. Canola, Canadian turnip, rutabaga (1999) Turnip rape, turnip green, field mustard, Brown et al. (1999), and Ulmer et al. Brassica rapa L. (=B. compestris (L.)) canola (2002) Brassica carinata L. Ethiopian mustard Ayalew et al. (2004) Brassica juncea (L.) Indian mustard, brown mustard Bodnaryk (1997), and Brown et al. (1999) Brassica napa L. Turnip Abro et al. (1994) Brassica nigra (L.) Black mustard Idris and Grafius (1996) Idris and Grafius (1996), and Badenes- Brassica oleracea L. var. acephala Collard, flowering kale Perez et al. (2004) Brassica oleracea L. var. alboglabra Kale Talekar and Shelton (1993) Idris and Grafius (1996), and Reddy et al. Brassica oleracea L. var. botrytis Cauliflower (2004) Abro et al. (1994), and Idris and Grafius Brassica oleracea L. var. capitate Cabbage (1996) Brassica oleracea L. var. gemmifera Brussels sprouts Talekar and Shelton (1993) Brassica oleracea L. var. gongylodes Kohlrabi Reddy et al. (2004) Idris and Grafius (1996), and Reddy et al. Brassica oleracea L. var. italic Broccoli (2004) Brassica rapa L. var. pakchoi Pak choi Talekar and Shelton (1993) Talekar et al. (1994), and Liu and Jiang Brassica rapa L. var. pekinensis Chinese cabbage (2003) Raphanus sativus L. Radish, bier radish Abro et al. (1994) Sinapis alba L. (=Brassica hirta Moench) White mustard, yellow mustard Bodnaryk (1997), and Brown et al. (1999)

Table 1.2 continued

(ii) Wild cruciferous host plants of P. xylostella Species Ratzka et al. (2002)s, and Barker et al. Arabidopsis thaliana (L.) Heynh Thalecress, mouse-earcress (2004) Idris and Grafius (1996), Shelton and Nault (2004), and Badenes-Perez et al. Barbarea vulgaris (L.) R. Br. Yellow rocket, rocketcress (2004) Berteroa incana L. DC Hoary alyssum Idris and Grafius (1996) Capsella bursa-pastoris (L.) Shepherd's purse, mother's-heart Idris and Grafius (1996) Cardamine flexuosa With. Flexuous bittercress Muhamad et al. (1994) Descurainia sophia (L.) Flixweed Talekar and Shelton (1993) Renwick and Radke (1990), and Idris and Erysimum cheiranthoides L. Wormseed mustard, treacle mustard Grafius (1996) Lepidium campestre (L.) R. Br. Field pepperweed Idris and Grafius (1996) Muhamad et al. (1994), and Begum et al. Lepidium virginicum L. Virginia pepperweed, peppergrass (1996) Raphanus raphanistrum L. Wild radish, wild rape, wild turnip Idris and Grafius (1996) Muhamad et al. (1994), and Begum et al. Rorippa indica (L.) Hiern Indian marshcress (1996) Rorippa islandica (Oeder) Barbàs Marsh yellowcress Muhamad et al. (1994) Sinapis arvensis L. (=Brassica kaber (DC) Wheeler) Wild mustard, crunchweed Idris and Grafius (1996) Sisymbrium altissimum L. Tumbling mustard, tall hedge mustard Talekar and Shelton (1993) Thlaspi arvense L. Stinkweed, pennycress, Frenchweed Idris and Grafius (1996)

(iii) Non-cruciferous plants on which P. xylostella is known to survive/develop Common Species Family Glucosinolates name Reference(s) Tropaeolum majus L. Tropaeolaceae Yes Nasturtium Renwick and Radke (1990) Cleome species Capparidaceae Yes Spider plant M. Sarfraz et al. (2005) P. Gupta and Thorsteinson (1960), Lohr (2001), and Löhr and Gathu Pisum sativum L. Fabaceae No Peas (2002) Hibiscus esculentis L. Malvaceae No Okra J. Gupta (1971)

1.2.4 Pest management

The global standard practice the use of synthetic chemical insecticides to control P. xylostella, but as a result of heavy and repeated use, the P. xylostella has become resistant to almost all insecticides (APRD, 2012; Furlong et al., 2013; Muhammad et al., 2005; Ridland & Endersby, 2011). However, P. xylostella rapidly evolves resistance to chemical insecticides, and was the first agricultural pest to develop field resistance to Bacillus thuringiensis (Bt) toxins (Ankersmit, 1953; Johnson, 1953; Talekar & Shelton, 1993), to which resistance is now widespread. P. xylostella was the second-ranked in exhibiting resistance to a high number of insecticide in the Arthropod Pesticide Resistance Database (APRD, 2012). The APRD listed 95 compounds for which P. xylostella has been reported to show resistance.

The rapid build-up of insecticide resistance has been seen mainly in tropical countries, where the over use of insecticides aims to control up to 20 P. xylostella generations in brassica crops per year (CABI, 2016). Although farmers have used insecticides for over 30 years in P. xylostella control, resistance to existing insecticides and the lack of new insecticides has led researchers to look for alternative control measures (CABI, 2016). Moreover, the use of non-selective insecticides leads to the destruction of natural enemies (Furlong et al., 2004). In response, integrated pest management (IPM) strategies have been developed. These strategies include a combination of chemical, biological and cultural control methods with an emphasis on maintaining natural enemies (Sarfraz et al., 2006).

The basic aim of a sustainable IPM program for P. xylostella is the introduction and conservation of natural enemies, which play a major role in limiting P. xylostella population growth (CABI, 2016). The establishment of Diadegma semiclausum (Hymenoptera: Ichneumonidae) and the use of the bacterium B. thuringiensis (Bt) and its toxins in the highlands in Indonesia, Malaysia, Taiwan and Philippines (Ooi & Kelderman, 1979; Poelking, 1992; Sastrosiswojo & Sastrodihardjo, 1986; Talekar et al., 1992) are examples of successful, coordinated control of P. xylostella.

Additionally, a wide range of natural enemies including parasitoids, arthropods fpredators, viruses, microsporidia, pathogenic fungi and bacteria have been known to

attack P. xylostella, though only few of them with significant results on field usage. (Furlong et al., 2013; Sarfraz et al., 2005) (Figure 1.7). Research over the past two decades has focused on manipulation and use of these organisms, particularly in classical biological control programs (see below) (Furlong et al., 2013). The use of entomopathogens, such as bacteria, virus, fungi and nematodes to control P. xylostella has been reviewed by Wilding (1986) and more recently by Cherry et al. (2002), and identified a number of pathogens infecting P. xylostella.

Figure 1.7 Major biological control agents of P. xylostella (Sarfraz et al., 2005).

Classical biological control

Classical biological control is the practice of importing and releasing natural enemies, to control an introduced pest (Hoffmann & Frodsham, 1993). It is used when an insect pest is introduced into another geographic area without its natural enemies (Waterhouse & Sands, 2001). The success of this application requires the correct identification of the natural enemy and its host strain (Sarfraz et al., 2005). Regions around the world where the majority of agriculture is based on introduced crops, in particular Australia, are notable for the high proportion of their exotic arthropod pests and the success of classical biological control (Waterhouse & Sands, 2001).

Australia’s first involvement with classical biological control was in 1888 and 1891 when A. Koebele visited Australia to find sources of parasitoids and predators of insect

pests that had become established in California and Hawaii (Waterhouse & Sands,

2001). Three parasitoids, D. semiclausum, Cotesia plutellae and D. collaris, have been introduced and then established successfully throughout Australia for control of P. xylostella (Sarfraz et al., 2005).

The first step in classical biological control is to determine the origin and identity of the introduced pest and then to collect appropriate natural enemies associated with it or with closely-related species. The natural enemy is fully described and then subjected to a rigorous quarantine process, to ensure that no unwanted organisms are introduced. It is then reared in large numbers and assessed for risk, with particular attention to host specificity (Hoffmann & Frodsham, 1993). The impact on non-target and native species in Australia is of particular importance in introduction of biological controls under the biosecurity act 2015, including in the introduction and registration of potential biopesticides based on microorganisms (Hauxwell et al., 2010).

Studies following release are conducted to determine if the natural enemy has successfully established at the site of release, and to assess the long-term benefit of its presence (Hoffmann & Frodsham, 1993). There are also situations in which biological control species have been introduced into quarantine but not liberated due to reasons such as poor breeding, lack of efficacy against the target host and/or lack of specificity (Waterhouse & Sands, 2001). When classical biological control is used, it is a requirement to ensure that only the target pest species is affected by the introduction of the natural enemy (Waterhouse & Sands, 2001).

Baculoviruses are invertebrate-specific virus pathogens that are used as efficient biopesticides (Asser-Kaiser et al., 2007; Hunter-Fujita et al., 1998). They are widely used in Australia due to their efficacy, relative stability (they can withstand being sprayed using standard farm equipment), high host specificity and lack of non-target infection in invertebrates (Hauxwell et al., 2010).

Plutella xylostella granulovirus (PlxyGV) of P. xylostella was first reported in Japan (Asayama & Osaki, 1969). Since then several scientists have reported the potential of granulovirus for use as a biological control agent for P. xylostella in Taiwan, India, Kenya, China and South Africa (Abdulkadir et al., 2013; Bin Abdul Kadir et al., 1999;

Grzywacz et al., 2001; Kadir, 1986; Rabindra et al., 1997). As Australia have not used this biological control yet, PlxyGV is being investigated as a potential biological control by QUT (Spence et al., 2016).

1.2.5 Taxonomy

Reliable systematics is necessary to understand taxa of pest species, the traits important to their management and host range, and to biological control (Cho et al., 2008). The morphology of genitalia was identified as an important and significant source of phylogenetically informative morphological characters (Beutel & Kristensen, 2012). Description of morphology of insect genitalia is now recognised as an essential standard for species-level taxonomy and inference of phylogenetic relationships (Schmidt, 2012).

The early classification of P. xylostella was described using morphological characteristics such as genitalia, wing venation, larval and pupal morphology, pupal and external morphology of adults (Clarke, 1971; Dugdale, 1973; Moriuti, 1986). Later, studies described more detailed morphological characters including character mapping (Baraniak, 2007; Robinson & Sattler, 2001) and one study examined the measurements of external morphological features of Indian P. xylostella populations (Chacko & Narayanasamy, 2004). However variation and plasticity in the morphological characters make it difficult to rely solely on these features and can result in an unsatisfactory phylogenetic resolution (Beutel & Kristensen, 2012). More recently, molecular sequence and integration of both molecular and morphology have been used to resolve the relationship of Plutella populations around the world. Similar approaches have been used combining both morphology and molecular data to describe wood white butterfly (Shtinkov et al., 2016), fruit flies (Schutze et al., 2014), spruce budworm (Lumley & Sperling, 2010) and Niganda moths (Pellinen & Wahlberg, 2015) to clarify their species status.

Molecular marker selection

Molecular systematics is used to infer the phylogenetic relationships among organisms using molecular data (DNA and RNA) to resolve competing hypotheses of species relationships and the placement of taxa whose relationships are known to be problematic (Judd et al., 1999). Caterino et al. (2000) stated that with sufficient specimens sampling the relationship among subspecies, species and species groups can be resolved with significant new insights. Similarly, Springer et al. (2001) pointed out that the value of mitochondrial sequences in phylogenetic analyses is further enhanced if they are collected in conjunction with nuclear sequences, because mitochondrial sequences provide an independent estimate of phylogenetic relationships that can be compared with estimates based on nuclear sequences.

Selecting a suitable marker is important for the accuracy in resolving species status (Rach et al., 2017). Since the introduction in 2003, the mitochondrial CO1 barcode gene region (cytochrome c oxidase 1) has become the most widely used molecular marker among most of the animal phyla (Kress et al., 2015).

Higher mutation rates and more rapid sorting of variation usually results in divergence of mtDNA sequences among species and a comparatively small variance within species, and the use of CO1 barcode relies on the principle that genetic variation within species is smaller than that between species (Lukhtanov et al., 2009). The estimated rate of divergence at the CO1 locus in insects was reported as 2% per million years (Brower, 1994; Juan et al., 1995).

COI has been widely used for DNA barcoding of insects (Hebert et al., 2003). DNA barcoding was proposed as an inexpensive and effective method to identify living organisms using an approximately 658 bp of mitochondrial DNA (mtDNA) as a barcode. Several insect studies have used the mitochondrial cytochrome oxidase 1 marker to study taxonomy, population and evolution (Chang et al., 1997; Landry et al., 1999; Miller et al., 2015; Schmidt et al., 2015). It has been also reported that CO1 barcode based delimitation of species is a good start for taxonomic processes (Hebert et al., 2004).

Mitochondrial DNA (mtDNA) markers are considered to be more sensitive than nuclear markers for population genetic studies (Loxdale & Lushai, 1998). Mitochondrial genes in general have several advantages such as the ability to amplify easily due to the high copy numbers per cell and their haploid character. They also evolve much faster than the coding regions of nuclear genes because mitochondria lack a proofreading mechanism (Rach et al., 2017). However, reliance on mitochondrian sequences and in particular on the COI barcode alone has been questioned. Mitochondrial DNA alone cannot provide good results in defining new species because of factors such as reduced effective population size and introgression, maternal inheritance, recombination, inconsistent mutation rate, heteroplasmy and compounding evolutionary processes, and COI sequence data should be used in conjunction with nuclear DNA, morphology, or ecology (Rubinoff et al., 2006; Rubinoff & Holland, 2005).

Integrative Taxonomy

Species definition has now become a highly-debated topic in modern systematics where new molecular species delimitation methods are being developed (De Queiroz, 2007; Morando et al., 2003; Pons et al., 2006; Puorto et al., 2001; Templeton, 2001; Wiens & Penkrot, 2002). Integrative taxonomy is the use of many different sources of data such as molecular, morphological, behavioural and ecological data to delimit species in a reliable manner (Dayrat, 2005; Padial et al., 2010). Although the selected species concept will influence the choice, analysis and interpretation of data, to obtain a good outcome at least three sources of data or ‘disciplines’ must be used for more rigorous species delimitation hypotheses (Schlick-Steiner et al., 2010). A number of studies have been conducted under the integrative taxonomic framework to resolve species status such as the nematode species, Malagasy tree frogs, spruce budworm and harvestmen, all with promising results (Fonseca et al., 2008; Glaw et al., 2010; Lumley & Sperling, 2010; Wachter et al., 2015).

1.2.6 A new Plutella taxon in Australia

The Australian Plutellidae contain 26 species in 8 genera, of which several genera, such as Diathryptica Meyrick and Tritymba Lower, are endemic (Nielsen et al., 1996).

The genus Plutella was thought to be represented in Australia by a single introduced taxon, P. xylostella, until the description of two genetically divergent lineages of this taxon by Landry and Hebert (2013). Their initial study objective was to identify the Australian lepidopteran fauna using the CO1 barcode, as a result of which they described a new ‘Australian’ taxon, Plutella australiana, based on 8.6% sequence divergence in the barcode region of the mitochondrial CO1 gene and differences in the morphology of genitalia. The collections and analysis of Plutella taxa in the study were based on light trapped material and museum specimens from the Australian Capital Territory (ACT), New South Wales (NSW), South Australia (SA) and Queensland (QLD). On the basis of these samples, P. australiana was reported to be broadly distributed in the eastern half of Australia (Figure 1.8) (Landry & Hebert, 2013). However, their study did not include any larval collections, leaving the information on its host plant and its pest status unknown, and sampling is required to confirm the presence or absence of P. australiana in other parts of Australia and the wider region.

Figure 1.8 Sites in Australia where specimens of P. xylostella (red) and P. australiana (blue) have been collected. The circles show the proportion of the two species at each site. These records only include specimens identified through DNA barcode analysis (Landry & Hebert, 2013).

COI sequence data showed a clear difference between the two Plutella taxa, but P. australiana cannot be differentiated from P. xylostella using external morphology. Genitalia morphological characteristics of both females and males between the two taxa were described, but based on only a few slide preparations (5 P. australiana males and 2 P. australiana females) (Landry & Hebert, 2013).

A few records had previously suggested some evidence for the possible presence of a second taxon or variant of P. xylostella in Australia. An allozyme study of P. xylostella from 14 locations worldwide included specimens from five different locations in Australia (Adelaide, Brisbane, North Queensland, Melbourne and Sydney) and found significant differences among the samples from Australia (Pichon et al., 2006). Similarly, Roux et al. (2007) using inter sample sequence repeat (ISSR) marker showed genetic differences between P. xylostella populations from Melbourne and Sydney. In the region, the description of a curved tubular projection similar to that of P. australiana females was previously described by Dugdale (1973) from a single female specimen presumed to be P. xylostella collected from New Zealand in comparison to that of P. antiphona Meyrick 1901 and P. sera Meyrick 1886 (Figure 1.9). This may suggest the presence of P. australiana in New Zealand, but its presence in the region needs further examination.

Figure 1.9 Picture from Dugdale (1973) showing the curved tubular projection (12) in a female genitalia from a diamondback moth specimen collected from New Zealand.

As an overall summary, the description of P. australiana in Australia has left many questions to be answered such as its species status, are the morphological features reliable when applying it to a wide range of specimens of the two taxa, distribution in other part of Australia and other countries especially in New Zealand, host plants and pest status and in particular the implications for the Australian pest management. The introduction of the new taxon requires additional testing for proposed biological controls, including biopesticides.

This thesis aims to broaden the knowledge of the description and distribution of new taxon, P. australiana, including its potential threat to Australian brassica production,

and to identify morphological features that can be used to differentiate P. xylostella and P. australiana. The results contribute to the better understanding of possible risks of the new taxon to Australian brassica production, and to inform potential pest management strategies.

Chapter 2: Molecular and morphological examination of Plutella species in Australia and New Zealand

2.1 Introduction

The diamondback moth (DBM), Plutella xylostella L. (Lepidoptera: Plutellidae), was first reported as an introduced species to Australia in 1882 (Tryon, 1889), and has since become widespread (French, 1893; Thompson & Moore, 1895) (Figure 2.1).

Figure 2.1 Distribution of diamondback moth in Australia. Blue = widespread, black = present (CABI, 2016).

In 2013, Landry and Hebert described a new taxon, Plutella australiana, in Australia. The study examined specimens collected using light traps between 2004-2012 from the Australian Capital Territory (ACT), New South Wales (NSW), South Australia (SA) and Queensland (QLD) and pinned museum specimens. The taxon description was based on an analysis of the mitochondrial cytochrome oxidase 1 (CO1) ‘barcode’ gene sequence (Figure 2.2). The new taxon was described as being broadly distributed in southern and eastern Australia. Further sampling was required to confirm the presence or absence of P. australiana in other parts of Australia, specifically Tasmania, where there was no record of occurrence.

Figure 2.2 Neighbor joining tree (nodes collapsed) based on Kimura-2-parameter distances for the barcode region of the cytochrome c oxidase 1 gene. Specimens are labelled by the Australian state or by country of origin and bracketed numerals indicate the number of specimens from each site (taken from Landry and Hebert (2013)).

The genital morphology of P. xylostella has been described (Baraniak, 2007; Dugdale, 1973; Landry & Hebert, 2013; Moriuti, 1986; Robinson & Sattler, 2001) but the description of P. australiana genital morphology is based on only a few specimens (Landry & Hebert, 2013): five P. australiana males and two P. australiana females. It has been suggested (Personal communication from John Dugdale, Senior entomologist, New Zealand) that the morphology of some New Zealand female specimens based on Landry and Hebert (2013) genitalia descriptions, might indicate the presence of P. australiana, but this has not been confirmed using CO1 sequence data. Those specimens were from early 19th century (example: 1907). However, as the description of P. australiana genitalia were based on few samples there is a need to further examine and identify the reliable diagnostic genitalia morphological features using a larger sample number of both taxa.

The described new ‘native’ Australian taxon closely related to P. xylostella, would potentially be at risk from biological control agents released against the introduced pest, and thus prevent or delay release or registration of new biological controls that are essential to an integrated resistance management strategy. Further work is therefore required to determine the taxonomic status of P. australiana, its geographic distribution and potential economic importance as a pest. The results using a larger sample size of both taxa will help to further clarify the identity of the two taxa, to identify the reliable diagnostic feature that can be used to identify both taxa and to expand the knowledge of P. australiana distribution in Australia.

This chapter presents the comparison of new adult specimens of P. xylostella and P. australiana collected from south-eastern Australia (including Tasmania) and from New Zealand. Mitochondrial CO1 sequence data was used to establish taxon identity which was then compared with a detailed morphological analysis of the genitalia of the two Plutella taxa including measurements of the genitalia characteristics to determine the statistical variance across taxa and populations along with an examination of morphological characteristics to identify reliable diagnostic features.

2.2 Materials and Methods

2.2.1 Sampling

Moths were collected using 40W ranger moth traps (Watkins and Doncaster, UK) connected to a 12V/15Ah valve regulated lead acid battery (Figure 2.3). New moon dates were considered when selecting the light trapping dates as moonlight is known to influence (decrease) monthly moth catch rates (Nowinszky et al., 1979; Williams, 1936). Collections were undertaken in Tasmania and eight different locations in eastern Australia (Queensland and New South Wales) and conducted between August to December in 2014 and 2015 and between May to October in 2016. Adult collections were sorted from light trap samples using an electric pooter (Australian Entomological Supplies) and transferred into solo cups or take away containers for transportation. Thirty individuals per site were selected for molecular analysis at each site except where less than thirty individuals were caught, in which case all the specimens collected were used. Specimens were also obtained from ten locations in New Zealand and from a laboratory reared colony. Samples from New Zealand were received preserved in 40% ethanol collected between 2005 and 2016 as both adults and larvae. Individuals from the lab reared colony and from Tuakau (2006) were not included in the analyses as good quality sequences could not be obtained at the beginning. A summary of the samples included in the analysis is shown in table 2.1.

Figure 2.3 A) Ranger moth light trap in a cabbage field and B) Insects trapped in the light trap. Moths of Plutella spp. are circled in yellow.

Table 2.1 Locations of DBM adult collections and number of individuals taken for genetic and morphological analyses. Specimens not taken for morphological analyses are marked as ‘/’ and unknown GPS coordinates are marked as ‘-‘.

Number of Number of specimens Date sequences used in used for morphological Sample code GPS Location (year) the phylogenetic identification analyses Females Males 27.34304 °S, SAM 2014 Samford, QLD 3 0 3 152.90909 °E 33.98295 °S, HOB3 2014 Hobart, Tasmania 16 4 7 150.63965 °E 33.98295 °S, SYDT 2014 Theresa Park, NSW 8 6 1 150.63965 °E 34.00338 °S, SYDW 2014 Werombi, NSW 10 10 / 150.56716 °E 27.493653 °S, BIR 2015 Birkdale, QLD 5 1 4 153.204733 °E 33.98295 °S, SYD15T and T 2015 Theresa Park, NSW 29 / 10 150.63965 °E 34.00338 °S, SYD15W 2015 Werombi, NSW 22 9 / 150.56716 °E 34.15285°S, M15 2015 Mowbary Park, NSW 13 11 / 150.55449°E 27.537202 °S, G 2016 Gatton, QLD 5 / / 152.335257 °E 27.754827 °S, L 2016 Laidley, QLD 4 / / 152.369293 °E DBM1 2016 - Nelson, New Zealand 14 5 5 Hobson, Rakaia, New DBM2 2016 - 3 / / Zealand Hobson, Rakaia, New DBM3 2016 - 7 / 7 Zealand DBM4 2013 - Pukekohe, New Zealand 29 4 5

DBM5 2013 - Levin, New Zealand 10 4 6

DBM6 2014 - Lincoln, New Zealand 3 / /

DBM9 2008 - Southbridge, New Zealand 23 5 5

DBM10 2008 - Chertsey, New Zealand 12 7 1

2.2.2 Molecular analysis

DNA extraction, PCR amplification and sequencing In total 216 adults were DNA extracted and Sanger sequenced (this sample number represents only the specimens with more than 75% sequence quality). DNA was extracted using the ISOLATE II Genomic DNA Kit (Bioline, Australia) from one leg of each specimen and with an overnight incubation at 56 ˚C. The 658 bp fragment of mitochondrial CO1 was amplified using primers LepF1 (5’- ATTCAACCAATCATAAAGATATTGG-3') and LepR1 (5’- TAAACTTCTGGATGTCCAAAAAATCA-3’) (DeWaard et al., 2007). PCR amplification was carried out with 10.5 µl 10% Trehalose, 2.625 µl of 5X buffer (Bioline), 0.125 µl of MyTaq HS Red DNA Taq polymerase (Bioline), 0.25 µl of each primer, 6 µl of template DNA, 1.25 µl of 25 mM MgCl2 (Bioline), and made up to a final volume of 25 µl with deionized water (ddH2O). Amplifications were performed in an Eppendorf Mastercycler® Pro S thermal cycler with an initial denaturing step at 94 ˚C for 2 minutes, followed by 45 cycles at 94 ˚C for 40 seconds, 54 ˚C for 40 seconds, 72 ˚C for 1 minute. PCR products were separated in 1.5% agarose gel using TBE buffer (40 mM Tris-acetate, 1 mM EDTA) for 50 minutes at 90 volts to confirm the quality of the PCR product. PCR products were purified using the ISOLATE II PCR and Gel Kit (Bioline, Australia). Purified PCR product was amplified in a sequencing reaction containing 2.0 μl of PCR product, 1.0 μl of 3.2 pmol forward primer, 1 μl of ABI Prism® Big Dye Terminators version 3.1 (Applied Biosystems, California, USA), 3.5 μl of 5x sequencing dilution buffer adjusted to a total reaction volume of 20μL with ddH2O. The sequencing cycle protocol involved initial denaturing at 96 °C for 5 minutes, followed by 30 cycles of 96 °C for 10 seconds, 50 °C for 5 seconds, 60 °C for 4 minutes, before a final hold at 15 °C for 10 minutes. The purified products were eluted in 20 μl of Elution Buffer C (Bioline, Australia) and then cleaned using a standard ethanol precipitation protocol prior to sequencing using both forward and reverse primers, by the QUT Institute for Future Environments-Central Analytical Research Facility (IFE-CARF) or Macrogen Inc. (South Korea). Sequencing was performed using an AB 3500 Genetic Analyser in QUT IFE-CARF, while an automatic sequencer ABI3730XL was used in Macrogen Inc. (South Korea). Obtained sequence results were used for an initial identification by blasting (BLAST) against the available sequences in Genbank database using a 100% match.

Phylogenetic analyses Either the forward or reverse sequence with a quality score higher than 75% was used in the alignment. Low quality sequence ends were trimmed using a modified Mott algorithm (http://www.unipos.net/download/Geneious7Manual.pdf). Sequences were aligned using default alignment parameters in MUSCLE within Geneious version 9.0.4 (http://www.geneious.com, Kearse (2012)). Ambiguously aligned blocks were excluded from the analyses. Further adjustments were made by eye and gaps within the alignment were treated as missing data.

Two hundred and sixteen (n=216) sequences obtained from adult individuals and 204 sequences obtained from adults reared from field collected larvae mentioned in chapter 3 (section 3.2.2.) were included in the final dataset. Additionally, reference sequences from Genbank for both P. xylostella and P. australiana (n=12 each, shown with accession numbers in the phylogenetic tree, see Appendix A) and three outgroups [two closely related species; Plutella hyperboreella, Plutella porrectella and the closely related genus Hyperxena scierana were also included in the analysis (see Appendix A for accession numbers)].

Phylogenetic analyses were conducted using Bayesian and maximum likelihood methods using the CIPRES Science Gateway (Miller et al., 2010). The substitution model was set as TIM2+I as found using jModel test 2 (Darriba et al., 2012). The maximum likelihood (ML) analysis was performed using RAxML 8.2.8 (Stamatakis, 2006). Support values for ML trees were estimated with 1000 bootstrap replicates. Bayesian analysis was performed using MrBayes 3.2.6 (Ronquist et al., 2012). The Bayesian analysis was run for 10,000,000 generations with trees sampled every 1000 generations with the default nruns=2 and nchains=4. Trees were visualized using TreeGraph 2 version 2.13.0 (Stöver & Müller, 2010).

The sequence divergence between the two taxa was quantified using the Kimura 2 parameter in MEGA version 7.0.18 (Kumar et al., 2016).

2.2.3 Morphology

Preparation and identification using genitalia morphological features A subset of both male and female adults (n=120) from three Australian states (Queensland, New South Wales, Tasmania) and New Zealand were dissected for comparison of the genitalia. A total of 54 males and 66 females were examined, of which 25 males and 41 females were Australian specimens, and 29 males and 25 females were from New Zealand (see appendix B and C for measurement data).

Dissection of both female and male genitalia followed the method described in Landry (2007) with slight modifications. The abdomen was cut at the sixth abdominal segment in both males and females to separate the genitalia structure. The structure was then put into an eppendorf tube containing 20% KOH and macerated (softened or separated) in a hot water bath just below simmering point for 10 minutes. It was then transferred onto a microscopic slide containing distilled water with a drop of diluted dishwashing detergent to break the surface tension. While the male genitalia structure was carefully removed from the macerated abdomen segments, the female structure was carefully separated at the 7th abdominal segment (sternite 7/S7). Any scales and further remaining macerated tissues were removed by gently brushing with a fine tipped nylon artist brush in both male and females. The clean structures were then put into an eppendorf tube containing 100% ethanol until taken for measurements or slide preparation.

Genitalia structures were viewed using a Nikon eclipse 50i light compound microscope and images were captured using a Nikon digital camera attached to a Nikon compound microscope under 4x magnification and the NIS-Elements BR digital image analysis software. Measurements were taken using the latter mentioned software.

The clean structures were kept on a glass slide with a water drop mixed with diluted dishwashing detergent to break the surface tension, and kept at their lateral positions to be photographed and then measured. Lateral positions of the structures were used in both male and female preparations; in males because the concaved structure of the vinculum saccus causes them to turn to the side when in the ventral position, and the female structures in order to minimize the damage to the fragile structure.

The characteristic features described by Landry and Hebert (2013) and observed in this study are shown in tables 2.2 and 2.3. In females, the tubular projection was reported to be more curved in P. australiana, and the raised pair of folds forming surrounding the antrum were reported to form two conical projections in P. xylostella that are absent in P. australiana (Table 2.2). In males, the appearance of the vinculum saccus was reported to be more slender (Table 2.3), the ventral margin of the valva to have a slight sinuation and the ventro-distal margin to be rounded in P. australiana.

In addition, key features were measured. In males, the combined length of the valva and vinculum saccus as the whole length, the width and length of the valva, the length of the vinculum saccus and the length of the phallus (Figure 2.4A) were recorded. In females, the whole width and length, the length of the upper part (S8 including the ovipositor), the length of the tubular projection, and length of the sternite 7 (S7L) were recorded (Figure 2.4B).

Table 2.2 Morphological features of DBM female genitalia examined in this study to identify diagnostic features. Features are circled in red. Images were taken by Tharanga Kariyawasam. P. australiana P. xylostella Lateral view Lateral view

Tubular projection has a curved apical half. Tubular projection is straight and evenly broad.

Ventral view Ventral view

Abdominal sternum 7 has a flat surface Abdominal sternum 7 has a raised pair of folds surrounding the antrum. forming surrounding the antrum which forms two conical projections bracing the tubular projection of the antrum.

Table 2.3 Morphological features of DBM male genitalia examined in this study to identify diagnostic features. Features are circled in red. The ventral view of P. australiana and lateral views of the valva were taken from Landry and Hebert (2013). The remaining images were taken by Tharanga Kariyawasam.

P. australiana P. xylostella

Ventral view Ventral view

Lateral view Lateral view

Vinculum saccus is slender. Vinculum saccus is broader.

Lateral view Lateral view

Slight sinuation in the ventral margin Straight in the ventral margin (indicated by (indicated by the arrow). Rounded ventro- the arrow). Less distinctly angled ventro-distal distal margin (right). margin (right).

Figure 2.4 Measurements of both female and male adult genitalia characteristics recorded to determine the statistical variance across taxa (Scale bars = 200 μm). Images were taken by Tharanga Kariyawasam.

A) male: WL = whole length, VW = valva width, VL = valva length, PL = phallus length, VSL = vinculum saccus length.

B) female: WL= whole length, WW = whole width, UPL = upper part length, TPL = tubular projection length, S7L = sternite 7 length.

Statistical analyses Australian P. xylostella, Australian P. australiana and New Zealand P. xylostella specimens were compared as three separate populations. All data were tested for normality and homogeneity of variance using a Shapiro Wilk normality test and Levene’s test for equality of variances before being analyzed. One-way ANOVA was conducted to examine the differences of the mean of each parameter between the three populations. A post hoc Tukey Honest Significant Differences (TukeyHSD) test was used to assess significant differences between the populations. Statistically significant parameters were visualized using density plots. All statistical analyses were conducted using the statistical software R (R Development Core Team, 2013).

Statistical analysis of female genitalia Measurements of whole length (WL), upper part length (UPL), tubular projection length (TPL) and sternite 7 length (S7L) data were normally distributed, but whole width (WW) was not. Normal distribution of whole width data was obtained using a power of 3 (^3) transformation before analysis. One-way ANOVA was conducted to examine the differences of the mean of each parameter across the three populations. When ANOVA showed significant differences for any parameter, TukeyHSD test was used to assess the significant differences between the three populations. Statistically significant parameters were visualized using density plots.

Statistical analysis of male genitalia Measurements of whole length (WL), valva width (VW), valva length (VL) and vinculum saccus length (VSL) were normally distributed, but the phallus length (PL) was not. Transformation of the PL data to obtain a normal distribution was performed using a log10 transformation before analysis. One-way ANOVA was conducted to examine the differences of the mean of each parameter across the three populations. When ANOVA showed significant differences for any parameter, TukeyHSD test was used to assess the significant differences between the three populations. Statistically significant different parameters were visualized using density plots.

2.3 Results

2.3.1 CO1 sequence data

DNA sequencing of CO1 barcode region identified the presence of P. xylostella and P. australiana. Phylogenetic trees showed two distinct clades with 100% support values in both maximum likelihood and Bayesian trees (Figures 2.6 and 2.7).

Phylogenetic analyses using both maximum likelihood (ML, Figure 2.6) and Bayesian analysis (Figure 2.7) showed similar topologies, forming two distinct and well- supported clades (with 100% support value) for P. australiana and P. xylostella. Reference sequences from Genbank were well resolved between the two clades (see Appendix D and E). All outgroups were also well resolved.

While interspecific variation between the two Australian taxa was 9% the intraspecific variation within both Australian taxa was 1.3%.

Both taxa were found sympatrically in most locations, including Tasmania (Figure 2.5). Four collections (Samford 2014, Theresa Park 2014, Gatton 2016 and Laidley 2016) contained only P. xylostella. All New Zealand collections were P. xylostella (not shown in Figure 2.5).

100% 90% 3 1 80% 2 11 70% 18 60% 11 50% 3 8 5 4 40% 13 9 30% 3 11 20% 11 10% 2

0% Percentage of taxa in each collection each in taxa of Percentage

P. xylostella P. australiana

Figure 2.5 Distribution of DBM adults caught in light traps at Samford (QLD), Hobart (TAS), Theresa Park (NSW), Werombi (NSW), Mowbray Park (NSW), Birkdale (QLD), Gatton (QLD) and Laidley (QLD). Numbers within bars represent the individuals identified and assigned to relevant taxa (see Table 2.1. for total number of individuals).

Figure 2.6 Bipartition maximum likelihood (ML) tree with bootstrap values. The tree was collapsed to remove low supported nodes (≥75%) and the nodes were further collapsed (shape of the clade) because of the large number of specimens assigned to each taxon. See Appendix D for the original phylogenetic tree.

Figure 2.7 Bayesian analysis with posterior probabilities. The tree was collapsed to remove low supported nodes (≥75%) and the nodes were further collapsed (shape of the clade) because of the large number of specimens assigned to each taxon. See Appendix E for the original phylogenetic tree.

2.3.2 Morphology

The majority of key features observed were not consistently different between the two taxa examined in the current study. The only consistently diagnostic feature was found in females, in which the form of the tubular projection was consistently curved in P. australiana, and in P. xylostella the raised pair of folds forming surrounding the antrum consistently formed two conical projections bracing the tubular projection of the antrum. In females, these forms matched the identification by CO1 sequence in 100% of female specimens from all three populations.

In males, species identification using differences in the appearance of the vinculum saccus (which had been reported to be slender in P. australiana) were found to be unreliable diagnostic features. The appearance of the vinculum saccus showed variation across the taxa and included intermediate characteristics. Similarly, the slight sinuation in the ventral margin and the rounded ventro-distal margin of the valva showed variation across the two taxa (see Appendix F). Two individuals were found to have opposing results when comparing morphology to the CO1 data: in one, sequence data confirmed the species as P. xylostella but the vinculum saccus was slender; in the other, CO1 data identified P. australiana but the vinculum saccus was broad (Table 2.4). The CO1 sequences from these two individuals were re-examined; DNA from each specimen was re-extracted and PCR and sequencing was repeated. The morphology was also re-examined, which confirmed the observation. CO1 identification of the taxa were used for these two individuals when conducting the subsequent statistical analyses of morphometric data.

Table 2.4 Images show the appearance of the vinculum saccus in two males where one had the characteristic morphology of P. australiana but were identified as P. xylostella from CO1 sequence data and the other had the characteristic morphology of P. xylostella but were identified as P. australiana from CO1 sequence data. These specimens were collected from Hobart and Theresa Park respectively.

Sample ID Morphology Molecular Genitalia image (4x)

HOB3_3 P. australiana P. xylostella

SYD15T_22m_Oct P. xylostella P. australiana

Statistical analysis of female genitalia One-way ANOVA results showed that the mean length of the TPL and S7L were significantly different between the groups (Table 2.5). TukeyHSD results showed that the mean length of TPL was shorter in P. australiana (P < 0.001) than in either the New Zealand or Australian P. xylostella populations but the tubular projection length was not significantly different in the two P. xylostella populations. The mean length of S7L was shorter in P. australiana (P < 0.001) than in Australian P. xylostella population and shorter in New Zealand P. xylostella population than in Australian P. xylostella population. However, the sternite 7 length was not significantly different between the New Zealand P. xylostella population and the Australian P. australiana population.

However, density plots of both TPL and S7L for all three groups showed significant overlap of 94% and 82% respectively with values within the variance of the opposite

taxon (Figure 2.8). There was no significant difference in the whole length, whole width and upper part length among the three populations.

Table 2.5 Mean values ± SD of parameters for each female population are shown in micrometers (μm). P-value of the one-way ANOVA (at 0.95 confidence intervals) are presented which showed a significance difference for the TPL (tubular projection length) parameter and the S7L (sternite 7 length) parameter. Statistically significant codes: ***P < 0.001, **P < 0.01.

Parameter Populations P-value F-value

Australian Australian New Zealand P. australiana P. xylostella P. xylostella (AA) (n=14) (AX) (n=27) (NX) (n=25) WL 747.4 ± 86 760 ± 98.5 739.2 ± 74.5 0.702 0.356 WW 468.6 ± 59.9 464.9 ± 54.2 465 ± 38.8 0.895 0.111 UPL 351.3 ± 75.9 303.8 ± 87.2 321.4 ± 81.4 0.244 1.442 TPL 177 ± 20.6 210.1 ± 24.7 218.9 ± 24.2 7.59e-06 *** 14.34 S7L 396.2 ± 71.2 463.7 ± 58.6 417.8 ± 36.7 0.00103 ** 7.729

Figure 2.8 Density plots showing the distribution of measurements taken for a) tubular projection length (TPL) and b) sternite 7 length (S7L) parameters of females showing an overlap between Australian P. australiana (AA), Australian P. xylostella (AX) and New Zealand P. xylostella (NX).

Statistical analysis of male genitalia One-way ANOVA results showed that the mean length of the PL and VSL were significantly different between the groups (Table 2.6). TukeyHSD results showed that the mean length of PL was longer in P. australiana (P < 0.001) than in either the New Zealand or Australian P. xylostella populations, but the PL was not significantly different in the two P. xylostella populations. The mean length of VSL was longer in P. australiana (P < 0.05) than in New Zealand P. xylostella population. Vinculum saccus length of Australian P. xylostella was not significantly different from both Australian P. australiana and New Zealand P. xylostella. However, density plots of both PL and VSL for all three groups showed a significant overlap of 85% with values within the variance of the opposite taxon (Figure 2.9). There was no significant difference in the whole length, valva width and valva length among the three populations.

Table 2.6 Mean values ± SD of parameters for each male population are shown in micrometers (μm). P-value of the one-way ANOVA (at 0.95 confidence interval) is presented which showed a significance difference for the PL (phallus length) parameter and VSL (vinculum saccus length) parameter. Statistically significant codes: *P < 0.05, ***P < 0.001.

Parameter Populations P-value F-value Australian Australian New Zealand P. australiana P. xylostella P. xylostella (AA) (n=12) (AX) (n=13) (NX) (n=29) WL 1006.7 ± 38.9 989.6 ± 36.5 973.5 ± 54.5 0.148 1.986

VW 321.4 ± 24.1 307.4 ± 21.7 313.7 ± 24.5 0.342 1.096

VL 607.9 ± 26.4 616.7 ± 25.8 611.1 ± 38.7 0.798 0.227

PL 565.9 ± 30.6 498 ± 31.2 493.9 ± 22.2 1.8e-08 *** 26.46

VSL 396.9 ± 34 368 ± 45 362.4 ± 29.8 0.0235 * 4.061

Figure 2.9 Density plots showing the distribution of measurements taken for (a) phallus length (PL) and (b) vinculum saccus length (VSL) parameters of males showing an overlap between Australian P. australiana (AA), Australian P. xylostella (AX) and New Zealand P. xylostella (NX).

2.4 Discussion

The results overall confirm the separation of the two taxa based on mitochondrial (CO1) sequence data. Both maximum likelihood and Bayesian analyses showed similar topologies by forming two distinct well supported clades (100%) for P. australiana and P. xylostella confirming them as two separate taxa. Moreover, an interspecific divergence of 9% and intraspecific divergence of 1.3% were found, further confirming the separation of the two taxa. These results are consistent with the description in Hebert et al. (2003) that the value closer to 3% in the CO1 sequence divergence suggests the presence of a separate taxon (Hebert et al., 2003).

Both P. xylostella and P. australiana were found sympatrically in most locations, including Tasmania. Two collections in 2014 (Samford and Theresa Park) and two collections (Gatton and Laidley) in 2016 consisted only of P. xylostella adults. The presence of P. australiana in Tasmania has been suggested previously based on an observation of morphology (personal communication from Lionel Hill, Senior Entomologist, in Tasmania) and the presence of P. australiana in Tasmania was confirmed by CO1 sequence data and morphological comparison in this study. A single female specimen with pronounced curvature of the tubular projection (which was described as P. xylostella) may indicate the presence P. australiana in New Zealand (Dugdale, 1973). Further supporting it another two female specimens, one collected from Ivercargill, Boulder Bank, Nelson in New Zealand in 1907 and another from Fiordland collected before 1910 were observed to have morphological characteristics of P. australiana based on the S7 oval sclerotisation with no raised pair of folds and the antrum projection in the side view (curvature and tubular projection projecting for less than 1/3 its length beyond 7S) as described by Landry and Hebert (2013) (personal communication from John Dugdale, Senior Entomologist, New Zealand). However, specimens examined in this study did not support the presence of P. australiana in New Zealand.

The morphology of genitalia in P. xylostella and P. australiana have been previously described (Baraniak, 2007; Dugdale, 1973; Landry & Hebert, 2013; Moriuti, 1986; Robinson & Sattler, 2001) but comparative analysis of measurements have not been previously recorded. This is the first study to address this gap.

There were no significant differences in the measures or features in the New Zealand or Australian P. xylostella populations except the sternite 7 length which showed a significant difference. There were significant differences between the two taxa except for the vinculum saccus length in males which showed no significant difference.

The length of the tubular projection in females was significantly longer in P. xylostella (from both Australia and New Zealand) than in P. australiana. The length of the sternite 7 was shorter in P. australiana than in Australian P. xylostella and in New Zealand P. xylostella than in Australian P. xylostella. However, the extensive overlap in the variance does not support those as diagnostic features. Similarly in males, the presence of individuals with intermediate and opposing characteristics of the vinculum saccus does not support the use of morphological characteristics in general as a diagnostic feature in males. Examinations of the vinculum saccus feature to be slender, the sinuation at the ventral margin of the valva and the rounded ventro-distal margin in P. australiana as described in Landry and Hebert (2013) cannot be used in the identification of the two taxa.

The examination of the curvature of the tubular projection in P. australiana and the raised folds surrounding the antrum in P. xylostella were reliable diagnostic features in females, further supporting the separation of the two taxa. The observations of the three female specimens reported by Dugdale (personal communication) suggests that further examination of New Zealand populations should be conducted to identify if P. australiana is present.

Whole length, whole width, and upper part length in females and whole length, valva width, and valva length in males were found not to be statistically different in the two taxa. Examination of genitalia measurements in their ventral position may show a different outcome, but it is difficult to use the ventral position, especially when handling a large number of specimens.

An allozyme study on P. xylostella populations from 14 locations worldwide, including specimens from five different locations from Australia (Adelaide, Brisbane, Mareeba, Melbourne and Sydney), found that the Australian and Japanese populations were different from all other populations (Pichon et al., 2006). Using six microsatellite

loci, Endersby et al. (2006) found no genetic variation within Australian populations or between Australian and New Zealand P. xylostella populations. Similarly, Juric et al. (2017) found that the Australian population clustered with the New Zealand haplotypes. These results are further supported by the results of this study, which show very little mitochondrial divergence and no significant morphological variation in the structure of genitalia between Australian and New Zealand populations. These results suggest that Australian and New Zealand populations of P. xylostella represent one population, and is potentially a consequence of a single, rapid invasion to both countries (Chu, 1986; Saw et al., 2006; Talekar & Shelton, 1993). Furthermore, the results are consistent with previous observations on strong migration capacity of P. xylostella (Saw et al., 2006) and of a possible recent introduction of DBM to Australia, New Zealand and North America from a highly variable population from Europe (Juric et al., 2017).

This study has confirmed that P. xylostella and P. australiana can be separated into two taxa based on both mitochondrial DNA and some morphological data (examination of female morphological characteristics). However, the species status of P. australiana has not been confirmed. The use of mitochondrial DNA alone to identify or describe new species has been questioned (Rubinoff et al., 2006; Will et al., 2005; Will & Rubinoff, 2004). Examination of only a single, maternally-inherited gene (mtDNA/CO1) may be insufficient, and ideally bi-parentally inherited nuclear markers should be identified and compared in order to identify species boundaries (see chapter 4 for further discussion). In addition, a combination of multiple sources of data in an integrative taxonomic framework using specimens from a wide geographic range (Dayrat, 2005; Schlick-Steiner et al., 2010; Springer et al., 2001) and interbreeding studies are required to determine if P. australiana is a new species.

Chapter 3: Host plants and distribution of Plutella species in Australia

3.1 Introduction

The diamondback moth (DBM), Plutella xylostella L. (Lepidoptera: Plutellidae), is one of the most destructive insect pests of brassica crops worldwide, attacking cole crops (cabbage, cauliflower), oilseeds (canola, mustard) and root vegetables (radish, turnip) (Sarfraz et al 2005). Crop damage is caused by larvae that feed on the leaves or the head of crops like in cabbage and broccoli (Figure 3.1) or on the flowers and young pods of canola.

Since the introduction of P. xylostella into Australia, it has been reported to attack brassica vegetable crops and now considered to cause severe damages to the canola production (Endersby et al., 2004). Damage in canola, can be severe and is increasing as the area planted increases in South Australia (SA), Western Australia (WA), Victoria (VIC) and New South Wales (NSW) (Furlong et al., 2008; Gu et al., 2007; Perry et al., 2015).

Plutella xylostella has developed resistance to almost all insecticides including Bt (the toxin from Bacillus thuringensis) and was the first agricultural pest to develop resistance against DDT (Ankersmit, 1953; Atumurirava et al., 2011; Sun et al., 1986; Talekar & Shelton, 1993; Zhou et al., 2011). The rapid and widespread evolution of resistance highlights the need for an effective integrated resistance management strategy including both biological controls and biopesticides. However, the release of biological controls against the exotic P. xylostella in Australia has been made more challenging with the description by Landry and Hebert (2013) of a new, closely related taxon, Plutella australiana. Little is known about this new taxon, particularly its potential economic importance as a pest along with its distribution. If it is an endemic species, it could be at risk from the biological control agents released against P. xylostella. Moreover, rigorous risk assessment on host specificity and impacts on non-

target organisms must be taken prior to the release of any new biopesticides based on microorganisms and insecticides against P. xylostella (BiosecurityAct, 2015).

Figure 3.1 DBM larvae damaging A) cabbage leaf, B) head formation of red cabbage.

The availability of brassica host plants and the abundance of P. xylostella on them are critical determinants of the economic importance of this pest (Ahuja et al., 2010). Plutella xylostella occurs on brassica crops but also occurs on weedy brassicas such as wild mustard and wild radish (Sarfraz et al., 2006; Sarfraz et al., 2011). Both P. xylostella and associated parasitoids use them as host plants and as refugia when cultivated crops are damaged or prior to their cultivation (Kahuthia-Gathu et al., 2009; Sarfraz et al., 2006; Talekar & Shelton, 1993). The incidence of P. xylostella on native Australian brassicas is not well known but there are many native species of brassicas in Australia, including caper berries, Capparis spp. P. xylostella have been successfully reared on caperbush (Capparis sandwichiana: Brassicales, capparacea) in Hawaii (Robinson & Sattler, 2001). Reports of P. xylostella on crops other than brassicas are very rare, but with few incidences recorded on; okra (Abelmoschus esculentus: Malvales, Malvaceae) in 1971 in Ghana (FAO, 1971), chickpea (Cicer arietinum: Fabales, Fabaceae) and prickly Russian thistle (Salsola kali: Caryophyllales, Chenopodiaceae) in northern Russia (Reichart, 1919; Talekar et al., 1985). Reappearance of DBM on those crops were not reported ever since (Löhr & Gathu, 2002). But recent records on a strain of P. xylostella has been reported to feed on sugar snap peas (Pisum sativum: Fabales, Fabaceae) (Löhr & Rossbach, 2001; Rossbach et al., 2006). The reason for this host shift was due to the high P. xylostella damage caused in the cabbage field causing the P. xylostella to shift to the only

available nearby crop, the sugar pea (Henniges-Janssen et al., 2014). However, the alleles of the newly evolved DBM population surviving on sugar peas were not fixed, showing genetic variation in adaptation for suitable environments (Henniges-Janssen et al., 2011).

There is limited data on the larval host plants of P. australiana, and its potential pest status is only now being explored. Only three larvae of P. australiana have been previously reported on Lincoln weed (Diplotaxis tenufolia: Brassicales, Brassicaceae) in South Australia (Perry et al., 2015). There are now large scale studies on its pest status in canola in South Australia (Perry et al., 2015; Perry et al., 2017), but data on its incidence in vegetable brassicas have not yet been reported and nothing is known of the host plants in eastern Australia.

This chapter investigates the larval host plants of the Plutella species, especially P. australiana on crops and weeds by collecting the larvae from both brassica crops and weeds in the fields. Identification of larvae collected from the field used both cytochrome oxidase 1 ‘barcode’ (CO1) sequence analysis and rearing of larvae through to adult emergence in the laboratory, followed by dissection, examination and morphometric analysis of the genitalia. Moreover, the genitalia morphological measurements between adults reared from field collected larvae and the light trapped adults (in chapter 2) were examined to look at any morphological variations caused by rearing individuals under laboratory conditions.

3.2 Materials and Methods

3.2.1 Sampling

Larvae were collected by plant inspection and hand collection, by sweep netting and using beat sheets at the same locations and same dates in Queensland, New South Wales and Tasmania as those from which adults were collected by light trapping (see chapter 2, section 2.2.1).

Thirty larvae were collected from each crop or from weeds on the field margin on the same day as light trapping was conducted. Larvae were identified from their pale brown head capsule and green and segmented body. Host plants were recorded, and extra leaves were collected from the field to feed the larvae when under laboratory conditions. Larvae were transported in take-away containers lined with paper tissue, along with host plant material, and closed with a ventilated lid sealed using a nappy liner to prevent larvae from escaping. Thirty larvae collected from Nelson, New Zealand in January 2016 were received from New Zealand as 40% ethanol preserved larvae. The majority of larvae were then reared on the extra leaves collected from the field accordingly and if the diet was not enough organic cabbage was used to feed the larvae. The larvae were reared in temperature controlled cabinets at 20-23 °C and humidity at 65-70% under 12 h light: 12 h dark until pupation and adult emergence. Adults were then frozen and stored for molecular and morphological analyses. Larvae collected from Queensland in 2016 were used for molecular analysis without rearing to their adult stage as well as the larvae shipped in ethanol from New Zealand.

3.2.2 Molecular Analysis

DNA extraction, PCR amplification and CO1 ‘barcode’ Sanger sequencing were conducted following the same procedure described in section 2.2.2 (chapter 2) to establish the identity of the taxa. Two hundred and four (n=204) larvae were included (Table 3.1) in the phylogenetic analyses described in section 2.2.2. (chapter 2).

Table 3.1 Larvae collection details including location, crop type and number of individuals taken for genetic and morphological analyses. Specimens not taken for morphological analyses are marked as ‘/’ and unknown GPS coordinates are marked as ‘-‘.

Number of sequences Number of specimens Sample Date GPS Elevation Location Crop type included in the used for morphological code (year) phylogenetic analyses identification

Females Males 33.98295° S, HOB4 2014 150.63965° E 31m Hobart, Tasmania cauliflower, swedes 13 8 5 33.98295° S, TL 2015 150.63965° E 58m Theresa Park, NSW cabbage 28 9 5 34.00338 °S, WL15 2015 150.56716 °E 286m Werombi, NSW kale 22 5 5 34.00338 °S, WW15 2015 150.56716 °E 286m Werombi, NSW weed (field mustard) 28 6 4 27.32139°S, GL 2016 152.20061°E 98m Gatton, QLD cauliflower 15 / / 27.45113°S, LL 2016 152.22041°E 151m Laidley, QLD broccoli 29 / / 28.13129 °S, CL 2016 153.23248°E 244m Currumbin, QLD broccoli , kale 11 / / 27.493653 °S, BL 2016 153.204733 °E 10m Birkdale, QLD broccoli 31 / /

NL 2016 - - Nelson, New Zealand kale 27 / /

3.2.3 Morphology

Detailed morphological analysis including measurements of adult genitalia characteristics to determine the statistical variance across taxa and populations (chapter 2, Figure 2.4); and observations of morphological characteristics to identify reliable diagnostic features (chapter 2, Table 2.2 and 2.3) were performed. A subset of both male and female larvae (n=47) from the same individuals taken for the phylogenetic tree construction were included in the statistical analyses (represents only Hobart, Theresa Park and Werombi). A comparison was made of the measured features of the genitalia of the adults reared from larvae within and between the taxa and with adults of both taxa from light traps (chapter 2) to determine any effect of laboratory conditions and diets, on the morphological features of the laboratory reared individuals. A total of 19 adult males and 28 adult females reared from larvae collected in the field were measured (see Appendix G and H for measurement data).

Statistical analyses

Field collected larvae All data were tested for normality and homogeneity of variance using a Shapiro Wilk normality test and Levene’s test for equality of variances before being analysed. All of the data for both females and males were normally distributed except the vinculum saccus length (VSL) in males, which was transformed by raising to the power of 3 (^3) transformation to obtain a normal distribution before analysis. One-way ANOVA was conducted to examine the differences of the mean of each parameter between the Australian P. xylostella and Australian P. australiana populations. Statistically significant parameters were visualized using density plots. All statistical analyses were conducted using the statistical software R (R Development Core Team, 2013).

Statistical analysis between adults caught in light traps and adults reared from field collected larvae

P. xylostella female individuals Measurements of all parameters were normally distributed. A one-way ANOVA was conducted to examine the differences of the mean of each parameter across the three populations (Australian P. xylostella adults, New Zealand P. xylostella adults and P. xylostella larvae). When the ANOVA results showed significant differences for any parameter, a post hoc Tukey Honest Significant Differences (TukeyHSD) test was used to assess the significant differences between the three populations. Statistically significant parameters were visualized using density plots.

P. australiana female individuals Measurements of whole length (WL), upper part length (UPL), tubular projection length (TPL) and the sternite 7 length (S7L) data were normally distributed, but whole width (WW) was not. Although transformation of the WW data to obtain a normal distribution was performed using a log10, log, raised to the power of 3 (^3), took the ninth root (^1/9), raises the constant e to the power of WW (exp), found the absolute value (abs), the sine of WW (sin), square root of WW (sqrt), it did not show a normal distribution. A non-parametric Kruskal Wallis test was conducted to examine the differences of the mean of each parameter across the two populations; Australian P.

australiana adults and P. australiana larvae. Statistically significant parameters were visualized using density plots.

P. xylostella male individuals While measurements of whole length (WL) and phallus length (PL) were not normally distributed other parameters were normally distributed. Normal distribution of WL and

PL data were obtained using a power of 3 (^3) transformation and log10 transformation, respectively before analysis. A one-way ANOVA was conducted to examine the differences of the mean of each parameter across the three populations (Australian P. xylostella adults, New Zealand P. xylostella adults and P. xylostella larvae).

P. australiana male individuals Measurements of all parameters were normally distributed. A one-way ANOVA was conducted to examine the differences of the mean of each parameter across the two populations; Australian P. australiana adults and P. australiana larvae. Statistically significant parameters were visualized using density plots.

3.3 Results

3.3.1 CO1 sequence Data

The sequences of all larvae could be aligned completely with the two taxa described from light trapped adult sequences (chapter 2), and all the samples (n=204) were fully resolved into either of the two taxa.

While the average interspecific variation between the two Australian taxa was 8.3% the intraspecific variation within P. xylostella and P. australiana were 1.3% and 0.3% respectively.

3.3.2 Host plants of P. xylostella and P. australiana

The majority of the larvae collected (n=160) were P. xylostella: 44 were larvae of P. australiana. The majority of collections were entirely composed of P. xylostella

(Figure 3.2). All New Zealand collections were P. xylostella (not included in Figure 3.2).

100% 90% 80% 70% 60% 22 22 50% 13 22 15 29 11 31 40% 30% 20% 10% 6 6

0% Percentage collection the taxa in of Percentage

P. xylostella P. australiana

Figure 3.2 Distribution of larvae from field collections in Hobart (TAS), Theresa Park (NSW), Werombi (NSW), Gatton (QLD), Laidley (QLD), Currumbin (QLD) and Birkdale (QLD). Numbers within bars represent the individuals identified and assigned to relevant taxa (for total numbers see Table 3.1.).

Larvae of P. australiana were present in only two collections: on cabbage in Theresa Park, NSW in 2015 and on field mustard weeds (Brassica rapa) amongst a kale crop in Werombi NSW in 2015 (Figure 3.2). Larvae collected from all other brassica crops were entirely composed of P. xylostella, including the New Zealand larvae. In the two collections in which P. australiana was present, they were sympatric with P. xylostella larvae, but the majority of larvae analysed from those two collections were P. australiana (22 out of 28 in both collections).

The larvae collected from the weed (B. rapa) on the margin of the kale crop at Werombi were predominantly P. australiana. Both larvae and pupae were found on the plants (Figure 3.3). However, larvae collected from the crop itself were entirely P. xylostella.

Figure 3.3 Field mustard (B. rapa) (above) and the cabbage field (below) that P. australiana larvae were collected. In order from left to right are the field mustard plant, its flower, larvae feeding on the leaf, pupae found on the stem and the cabbage field where P. australiana larvae were collected.

3.3.3 Morphology

As in chapter 2, the majority of observed morphological features of the genitalia used to differentiate the two taxa were found not to be consistently reliable in adults reared from larvae collected in the field. The two consistent diagnostic features were again found in females, in which the form of the tubular projection was consistently curved in P. australiana, and in P. xylostella the raised pair of folds surrounding the antrum consistently formed two conical projections bracing the tubular projection of the antrum. As for the adults caught in light traps (chapter 2), identification of the taxon using these two features matched the identification by CO1 sequence in 100% of female specimens.

In males, species identification using differences in the appearance of the vinculum saccus, which had been reported to be slender in P. australiana, the sinuation at the ventral margin of the valva and the rounded ventro-distal margin in P. australiana (Landry & Hebert 2013) were found to be unreliable diagnostic features. In one male adult reared from larvae, the morphological appearance of the vinculum saccus directly contradicted the CO1 sequence data. The individual was confirmed as P. xylostella by sequence data but the appearance of the vinculum saccus was slender (Table 3.2). The CO1 sequence from this individual was re-examined, with DNA re-extracted and subjected to PCR and Sanger sequencing, and the morphology was also re-examined. This re-confirmed the observation. The individual was categorised as P. xylostella in the statistical analysis of morphometric data.

Table 3.2 The image shows the appearance of the vinculum saccus of a male that had the characteristic morphology of P. australiana but was identified as P. xylostella from CO1 sequence data. The larva was collected from B. rapa weeds in Werombi NSW in 2015.

Sample ID Morphology Molecular Genitalia image (4x)

WW15_18 P. australiana P. xylostella

Statistical analysis of field collected larvae

Statistical analysis of female genitalia One-way ANOVA results showed that the mean of whole length (WL), whole width (WW), tubular projection length (TPL) and sternite 7 length (S7L) were significantly different between the taxa. The mean of WL in P. xylostella was longer (P < 0.01) than in P. australiana, WW in P. xylostella was wider (P < 0.05) than in P. australiana, TPL in P. xylostella was longer (P < 0.01) than in P. australiana and S7L in P. xylostella was (P < 0.01) longer than in P. australiana (Table 3.3). However, density plots of the WL, WW, TPL and S7L showed significant overlap of 64%, 68%, 54% and 61% respectively with values within the variance of the opposite taxon (Figure 3.4). The upper part length was not significantly different between the taxa.

Table 3.3 Mean ± SD values of parameters for each female population are shown in micrometres (μm). P-value of the one-way ANOVA (at 0.95 confidence intervals) is presented which showed a significance difference for the whole length (WL), whole width (WW) and tubular projection length (TPL) parameters. Statistically significant codes: *P < 0.05, **P < 0.01.

Parameter Populations P-value F-value

P. xylostella (n=15) P. australiana (n=13) WL 814.6 ± 80 747.2 ± 35.9 0.00958 ** 7.822 WW 485.6 ± 46.3 448.5 ± 16.9 0.0113 * 7.427 UPL 383.4 ± 68.4 385.4 ± 41.1 0.927 0.009 TPL 204.6 ± 42.1 159.7 ± 13.3 0.00105 ** 13.59 S7L 431.2 ± 81.5 361.8 ± 32.5 0.00799 ** 8.256

a) b)

c) d)

Figure 3.4 Density plots showing the distribution of measurements taken for a) whole length (WL), b) whole width (WW), c) tubular projection length (TPL) and d) sternite 7 length (S7L) parameters of females showing an overlap between P. xylostella and P. australiana. However, mean values are significantly different.

Statistical analysis of male genitalia One-way ANOVA results showed that the mean phallus length (PL) (P < 0.001) was significantly longer in P. xylostella than in P. australiana (Table 3.4). A density plot of PL showed an overlap of 11% with values within the variance of the opposite taxon (Figure 3.5). There was no significant difference in the whole length, valva width, valva length and vinculum saccus length between the two taxa.

Table 3.4 Mean ± SD values of parameters for each male population are shown in micrometres (μm). P-value of the one-way ANOVA (at 0.95 confidence intervals) is presented which showed a significance difference for the phallus length (PL) parameter. Statistically significant codes: ***P < 0.001.

Parameter Populations P-value F-value P. xylostella (n=12) P. australiana (n=7) WL 959.8 ± 39.5 961.5 ± 53.2 0.942 0.005 VW 316.2 ± 14.9 308.3 ± 18.5 0.348 0.931 VL 569.3 ± 27.2 606.2 ± 63.6 0.166 2.092 PL 552.2 ± 13.1 479.5 ± 17.3 1.12e-07 *** 81.48 VSL 390.5 ± 29.6 355.3 ± 54.6 0.133 2.485

Figure 3.5 Density plot showing the distribution of measurements taken for phallus length (PL) parameter of males showing an overlap between P. xylostella and P. australiana. However, mean values are significantly different.

Statistical analysis between adults caught in light traps and adults reared from field collected larvae

Female P. xylostella individuals One-way ANOVA results showed that the mean of whole length (WL), upper part length (UPL) and sternite 7 length (S7L) were weakly significantly different (P < 0.05) (Table 3.5). TukeyHSD results showed that the mean length of WL was shorter in New Zealand P. xylostella than in larvae. There was no significant difference between the WL of Australian P. xylostella and larvae; and New Zealand and Australian P. xylostella populations. The mean length of UPL was shorter in Australian P. xylostella than in larvae. There was no significant difference between New Zealand P. xylostella and larvae; and New Zealand P. xylostella and Australian P. xylostella. The mean length of S7L was shorter in New Zealand P. xylostella than in Australian P. xylostella. There was no significant difference between Australian P. xylostella and larvae; and New Zealand P. xylostella and larvae. However, density plots of the WL, UPL and S7L showed significant overlap of 94%, 91% and 93% respectively with values within

the variance of the opposite taxon (Figure 3.6). There was no significant difference in the whole width and tubular projection length among the three populations.

Table 3.5 Mean ± SD values of parameters for each female population are shown in micrometres (μm). One-way ANOVA (at 0.95 confidence intervals) is presented which showed a significance difference for the whole length (WL) and upper part length (UPL) parameters. Statistically significant codes: *P < 0.05.

Parameter Populations P-value F-value

Australian New Zealand Larvae adults (n=27) adults (n=25) (n=15) WL 760.0 ± 98.5 739.2 ± 74.5 814.6 ± 80 0.0307 * 3.687 WW 464.9 ± 54.2 465 ± 38.8 485.6 ± 46.3 0.335 1.112 UPL 303.8 ± 87.2 321.4 ± 81.4 383.4 ± 68.4 0.013 * 4.664 TPL 210.1 ± 24.7 218.9 ± 24.2 204.6 ± 42.1 0.305 1.212 S7L 463.7 ± 58.6 417.8 ± 36.7 431.2 ± 81.5 0.0235 * 3.989

a) b)

Figure 3.6 Density plots showing the distribution of measurements taken for a) whole length (WL), b) upper part length (UPL) and c) sternite 7 length (S7L) parameters showing an overlap between Australian P. xylostella adults (AUS adults), New Zealand P. xylostella adults (NZ adults) and P. xylostella larvae. However, mean values are significantly different.

Female P. australiana individuals One-way ANOVA and Kruskal Wallis test results (Table 3.6) showed that the mean whole width (WW) (P < 0.01) and the mean tubular projection length (TPL) (P < 0.05) were significantly different between P. australiana adults from the field and P. australiana adults reared from larvae. Both WW and TPL were shorter in adults from larvae than in adults from the field. However, both density plots of WW and TPL showed an overlap of 78% with values within the variance of the opposite taxon (Figure 3.7). There was no significant difference in the whole length, upper part length and sternite 7 length among the two populations.

Table 3.6 Mean ± SD values of parameters for each female P. australiana population are shown in micrometres (μm). One-way ANOVA (at 0.95 confidence intervals) and Kruskal Wallis test are presented which showed a significance difference for the whole width (WW) and tubular projection length (TPL) parameters. Statistically significant codes: *P < 0.05.

Parameter Populations P-value F-value

Adults from light Larvae traps (n=14) (n=13) WL 747.4 ± 86 747.2 ± 35.9 0.994 0 WW 468.6 ± 59.9 448.5 ± 16.9 0.01152* - UPL 351.3 ± 75.9 385.4 ± 41.1 0.176 1.943 TPL 177 ± 20.6 159.7 ± 13.3 0.0191 * 6.281 S7L 396.2 ± 71.2 361.8 ± 32.5 0.136 2.374

Figure 3.7 Density plots showing the distribution of measurements taken for whole width (WW) and tubular projection length (TPL) parameters of females, showing an overlap between Australian P. australiana adults from light traps and P. australiana adults reared from larvae. However, mean values are significantly different.

Male P. xylostella individuals One-way ANOVA results showed that the mean phallus length (PL) (P < 0.1) was weakly significantly different between adults caught in light traps including the New Zealand specimens, and adults reared from field collected larvae (Table 3.7). The mean of PL was longer in adults from larvae than in adults from the field. A density plot of PL showed an overlap of 93% with values within the variance of the opposite taxon (Figure 3.8). There was no significant difference in the whole length, valva width, valva length and vinculum saccus length among the three populations.

Table 3.7 Mean ± SD values of parameters for each male P. xylostella population are shown in micrometres (μm). One-way ANOVA (at 0.95 confidence intervals) is presented which showed a slight difference for the phallus length (PL) parameter. Statistically significant codes: ‘.’0.1.

Parameter Populations P-value F-value

Australian New Zealand Larvae adults adults (n=13) (NX) (n=29) (n=12)

WL 989.6 ± 36.5 973.5 ± 54.5 959.8 ± 39.5 0.159 2.135 VW 307.4 ± 21.7 313.7 ± 24.5 316.2 ± 14.9 0.913 0.012 VL 616.7 ± 25.8 611.1 ± 38.7 569.3 ± 27.2 0.588 0.302 PL 498 ± 31.2 493.9 ± 22.2 552.2 ± 13.1 0.084 . 3.263 VSL 368 ± 45 362.4 ± 29.8 390.5 ± 29.6 0.552 0.365

Figure 3.8 Density plot showing the distribution of measurements taken for phallus length (PL) parameter of males, showing an overlap between P. xylostella adults from light traps and P. xylostella adults reared from larvae.

Male P. australiana individuals One-way ANOVA results showed that the mean whole length (WL) (P < 0.05), valva length (VL) (P < 0.01) and the phallus length (PL) (P < 0.001) were significantly different between P. australiana adults from the field and P. australiana adults reared from larvae (Table 3.8). The mean of WL, VL and PL were shorter in adults from larvae than in adults from the field. However, density plots of WL, VL and PL showed an overlap of 58%, 58% and 42% respectively with values within the variance of the opposite taxon (Figure 3.9). There was no significant difference in the valva width and vinculum saccus length among the two populations.

Table 3.8 Mean ± SD values of parameters for each male P. australiana population are shown in micrometres (μm). One-way ANOVA (at 0.95 confidence intervals) is presented which showed a significance difference for the whole length (WL), valva length (VL) and phallus length (PL) parameters. Statistically significant codes: *P < 0.05, **P < 0.01,***P < 0.001.

Parameter Populations P-value F-value Adults from light Larvae (n=7) traps (n=12) WL 1006.7 ± 38.9 961.5 ± 53.2 0.0245 * 6.167 VW 321.4 ± 24.1 308.3 ± 18.5 0.612 0.267 VL 607.9 ± 26.4 606.2 ± 63.6 0.0073 ** 9.278 PL 565.9 ± 30.6 479.5 ± 17.3 0.000841 *** 17.29 VSL 396.9 ± 34 355.3 ± 54.6 0.691 0.164

Figure 3.9 Density plots showing the distribution of measurements taken for whole length (WL), valva length (VL) and phallus length (PL) parameters of males, showing an overlap between P. australiana adults from light traps and P. australiana adults reared from larvae. However, mean values are significantly different.

3.4 Discussion

The results of the larval collections confirm the separation of the two taxa based on mitochondrial (CO1) sequence as in chapter 2. The CO1 sequences were well resolved into P. australiana and P. xylostella. Moreover, an interspecific divergence of 8.3% and intraspecific divergence of 1.3% and 0.3% within P. xylostella and P. australiana respectively were found further confirming the separation of the two taxa as in chapter 2. The number of specimens of P. australiana considered in the analysis was not as high as for P. xylostella.

Since P. australiana was first described in 2013, very few larvae have been reported in the field, and those mostly on weeds. Three larvae were found on Lincoln weed (Diplotaxis tenufolia, family Brassicaceae) in South Australia (Perry et al., 2015). This study is the first report of wild field mustard, B. rapa, as a host for P. australiana larvae. P. australiana larvae are reported to attack canola, a cultivar of B. rapa or B. napus in industry reports (personal communication from Michael Keller, Professor, Adelaide) but the data has not yet been published.

This study has provided the first documentation of vegetable cabbage as a host for larvae of P. australiana. Plutella australiana appears to have little association with crop plants other than canola (as reported above). Larvae collected from cabbage, kale, broccoli, swede and cauliflower crops in Australia and analysed in this study were entirely composed of P. xylostella with the notable exception of the cabbage crop at Theresa Park NSW in 2015, in which both P. xylostella and P. australiana larvae were identified. The larvae collected from a kale crop in Nelson, New Zealand, were all found to be P. xylostella.

It was also noteworthy that while both P. australiana and P. xylostella larvae were identified in the B. rapa weeds on the margin of a kale crop at Werombi NSW, all the larvae analysed from the kale crop itself were P. xylostella. Furthermore, the results in chapter 2 show that at both these locations and sampling dates, a significant proportion of the adults caught in light traps were P. australiana. However, while the majority of larvae recovered from the cabbage crop at Theresa Park were P. australiana, at Werombi the larvae from the kale crop were only P. xylostella, despite the presence of

P. australiana as larvae on adjacent weeds and as adults in light traps. Similarly, the larval samples from cauliflower and swedes in Hobart, Tasmania were 100% P. xylostella but light trap samples from the same location were shown (in chapter 2) to have a small proportion of P. australiana (Figure 2.5).

Overall, these findings suggest that P. australiana may be an occasional or emerging pest of brassica crops, present only in some crops (canola, cabbage) but not yet widely attacking vegetable brassicas and possibly primarily found on weedy brassica species. Further investigation on a wider range of Brassicaceae plants is required which may reveal more host plants used by P. australiana.

The presence of P. australiana larvae on B. rapa, but not on kale in the same field, indicates a potentially significant host preference. Studies have found that P. xylostella choose their host according to the nutrient levels of host plants (Sarfraz, Dosdall, & Keddie, 2010) or even that they select host plants with less leaf wax (Stoner, 1990). The implications of potential host preference will be further discussed in chapter 4.

As in chapter 2, examination of the curvature of the tubular projection in P. australiana and the raised folds surrounding the antrum in P. xylostella were the only reliable diagnostic features in females. The other features measured were found to be statistically significantly different between the two taxa, but the overlap in the variance indicates that these features cannot be used as a diagnostic characteristic. In males, the presence of individuals with intermediate and contradictory appearance of the vinculum saccus further confirmed it as an unreliable feature, though both phallus length and vinculum saccus length were found to be significantly longer in P. australiana than in P. xylostella. As in chapter 2 observation of the sinuation of the ventral margin and the round ventro-distal margin of the valva were found not to be diagnostic due to their variation between the taxa.

Field collected larvae that were reared under laboratory conditions exhibited morphological differences in both females and males. Between light caught adults and larval females of P. xylostella, whole length and upper part length were found to be longer in larvae than in adults from both Australia and New Zealand. In P. australiana the whole length and tubular projection length were found to be shorter in larvae than

in adults. Between adult and larval males of P. xylostella, phallus length was found to be longer in larvae than in adults. In P. australiana, whole length, valva length and phallus length were found to be shorter in larvae than in adults. However, phallus length was found to be different between P. xylostella larvae and P. australiana larvae as mentioned above. These findings suggest that the length of some morphological features vary depending on rearing conditions.

In conclusion, the molecular results presented here further support the classification of P. australiana and P. xylostella into two taxa. However, morphological characters were found to be largely unreliable as a diagnostic tool, particularly for laboratory reared specimens except the diagnostic morphological features in females. The results of the field collections suggest that P. australiana may be an emerging pest of crops in Australia, but further studies are required to determine the full range of host plants used by this species and potential host plant preferences.

Chapter 4: General Discussion

This study investigated the taxonomy, distribution, host plants, pest status and the pest management implications of Plutella species in Australia including New Zealand. The main aim of this study was to clarify the validity of Plutella australiana as a distinct species by sampling individuals from different locations in Australia and a larger sample size by using a mitochondrial gene (CO1) and by using a detailed genitalia morphological analysis of the two Plutella taxa. Moreover, the distribution, pest status and management implications of these taxa were examined.

The key findings of this study were; • Plutella xylostella and P. australiana are two distinct taxa based on CO1 data. • The measurement of key morphological features, while significantly different, overlap and cannot be used as diagnostic tools in differentiating the two taxa. The examinations of the morphological features revealed only two reliable diagnostic features in females (the form of the tubular projection was consistently curved in P. australiana, and in P. xylostella the raised pair of folds surrounding the antrum consistently formed two conical projections bracing the tubular projection of the antrum). • Both taxa are found sympatrically in most locations in Australia and share similar host plants, but P. australiana are rarely found on vegetable brassicas. Preference of P. australiana for B. rapa suggests that canola crops widely grown in South Australia and Western Australia may be at risk. • Plutella xylostella was found in New Zealand but P. australiana was not. No significant differences in morphology between Australian and New Zealand P. xylostella were identified except the sternite 7 length which was longer in Australian P. xylostella than in New Zealand P. xylostella. Together, with the low genetic divergence found between the P. xylostella populations and strong migration capacity of P. xylostella, these data suggest a single widespread invasion to both countries or an invasion from New Zealand to Australia or vice versa. (Saw et al., 2006).

• The origin of P. australiana remains undetermined and this has implications for pest management strategies in Australia. Based on the reported estimated rate of divergence at the CO1 locus in insects (Brower, 1994; Juan et al., 1995) and the results of this study, P. xylostella and P. australiana diverged about 4.5 million years ago before even the introduction of P. xylostella into Australia. • No records have been found in the region, but the sequence diversity is low, suggesting a recent incursion. It is not clear if P. australiana evolved in Australia or elsewhere, or if it is a native or introduced taxon.

These findings are discussed below in relation to the existing knowledge on Plutella taxa in Australia including New Zealand.

4.1 CO1 ‘barcode’ analysis of Australian and New Zealand Plutella taxa

This study confirmed the separation of P. xylostella and P. australiana into two taxa using CO1 ‘barcode’ sequence data from both adults and larvae. Both maximum likelihood and Bayesian analyses showed similar topologies, forming two distinct well supported clades (100%) for P. australiana and P. xylostella, and confirming them as two separate taxa. Among adults the interspecific divergence of the CO1 ‘barcode’ sequence data between the two taxa was on average 9% while intraspecific variation within both taxa was 1.3%. Among larvae, the interspecific divergence of the CO1 ‘barcode’ sequence data between the two taxa was of average 8.3% while intraspecific variation within P. xylostella and P. australiana were 1.3% and 0.3% respectively, though the number of specimens of P. australiana was not as high as for P. xylostella.

Hebert et al. (2003) suggested that in lepidopterans a value closer to 3% in the CO1 sequence divergence represents a new species. The Landry and Hebert (2013) supports this, so the interspecific divergence of the current study (9% and 8.3%) shows P. xylostella and P. australiana as two distinct taxa. Similarly, Perry et al. (2017) showed a high genetic divergence between P. australiana and P. xylostella. These findings were consistent with other studies on mosquitoes (Ruiz-Lopez et al., 2012) and flies (Renaud et al., 2012) which have reported the existence of new taxa using CO1

barcode sequence divergence. However, the identity of P. xylostella and P. australiana as separate species, and their capacity to interbreed remains unresolved, requiring further molecular studies using nuclear markers and mating compatibility studies.

Many insect studies have used the mitochondrial cytochrome oxidase 1 marker to examine population genetics, taxonomy and evolution (Chang et al., 1997; Landry et al., 1999; Miller et al., 2015; Schmidt et al., 2015) due to its high genetic variability (Simon et al., 1994). However, there are arguments against using mitochondrial DNA alone in identifying or describing new species because it is haploid and maternally inherited with its rate of evolution inconsistent within and between species (Rubinoff et al., 2006; Will et al., 2005; Will & Rubinoff, 2004). A study of Phyciodes butterflies demonstrated that using mitochondrial DNA alone resulted in failure to correctly identify species (Wahlberg et al., 2003). Nuclear (RAD seq) markers have recently been used to identify different taxa (Perry et al., 2017) and may be a potential nuclear marker for future studies on Plutella taxa. However, mating compatibilities between P. australiana and P. xylostella also needs to be examined to confirm the species status of P. australiana.

The status of P. australiana as originating in and endemic to Australia is not clear. The estimated rate of divergence at the CO1 locus in insects was reported as 2% per million years (Brower, 1994; Juan et al., 1995). Based on this estimate and the results of this study (interspecific variation of 9% between the two taxa), P. australiana and P. xylostella diverged about 4.5 million years ago, prior to the introduction of P. xylostella approximately 120 years ago. The low level of variation of P. australiana CO1 sequences also suggests very limited gene flow among populations in Australia, and possibly recent introduction from elsewhere. Most Plutella spp. collections examined by Landry and Hebert (2013) were collected between 2004 and 2012. However, one museum pinned specimen examined, collected from NSW in 1971 by V. J. Robinson, was identified as P. australiana and therefore suggests the occurrence of P. australiana in Australia from at least that time.

A study using six microsatellite loci did not show any variation among Australian populations of P. xylostella across 17 locations (Endersby et al., 2006), and concluded

that the species had been recently introduced into Australia. However, Pichon et al. (2006) conducted a regional study of allozyme variation and found significant genetic differentiation among Australian populations of P. xylostella. Saw et al. (2006) examined haplotype frequencies and suggested that Australian populations may be the result of migration from northern regions of south-eastern Asia. Similarly, Talekar and Shelton (1993) suggested a possible migration of P. xylostella to the Southern Hemisphere including Australia. However, Juric et al. (2017) suggested a recent introduction of P. xylostella to Australia, New Zealand and North America from a highly variable European population and found that Australian population clustered with New Zealand haplotypes, and that two independent lineages were found to have arisen from Australia and New Zealand groups, one of them an entirely Australian haplotype.

Given the low levels of mitochondrial DNA divergence among populations of P. australiana found in the current study, it is unlikely that it is an Australian native species, but rather a recent introduction. Further examination of the phylogeography of P. australiana is required to determine if a migration pathway into Australia, similar to that of P. xylostella, has occurred or if it may have evolved in Australia.

4.2 Diagnostic morphological features

While Landry and Hebert (2013) proposed that a number of morphological characteristics could be used to distinguish the two Australian taxa, the majority of these features were found not to be reliable in differentiating adults of the two taxa. Two reliable diagnostic features to differentiate the taxa were found in females: examination of the curvature of the tubular projection in P. australiana and raised folds surrounding the antrum in P. xylostella. In males, species identification using differences in the appearance of the vinculum saccus (slender in P. australiana) was found to be an unreliable diagnostic feature. Similarly, the shape of the ventro-distal margin (rounded in P. australiana) and the slight sinuation in the ventral margin of P. australiana were found to be unreliable diagnostic features. Other features described by Landry and Hebert (2013) such as teguminal processes (ventral view), the zone of spiniform setae of the valva in males and the extension of the antrum projection beyond

the posterior margin of S7 (ventral view), ductus bursae in females still require further examination. While all the genitalia measurements in this study were taken in their lateral position, except the phallus in males, measurements of the features such as vinculum saccus width in males and whole width of sternite 7 in females in their ventral position would be interesting to examine if they give different results.

In males, the presence of individuals with intermediate and opposing characteristics of the vinculum saccus does not support the use of it as a diagnostic feature. This study found three male individuals (two field collected adults and one individual reared from larvae collected on field mustard) which showed opposing results for molecular and morphological identifications. These results seen only in males may suggest the possibility of mating compatibilities between the two taxa where P. australiana males mate with P. xylostella females and progeny produced have P. australiana genitalia morphology but P. xylostella mitochondrial DNA or vice versa.

Descriptions of morphological characteristics of Plutella species such as genitalia characteristics, wing venation, larval characteristics, pupal characteristics and adult external morphology of male and females (Clarke, 1971; Dugdale, 1973; Moriuti, 1986; Robinson & Sattler, 2001) have been used to identify Plutella species in other countries such as Hawaii and New Zealand. The observation on a curved tubular projection described by Dugdale (1973) in a single female collected from New Zealand suggests that P. australiana may be present in New Zealand but this requires further examination. Furthermore, Dugdale reported that the dark brown or blackish antennal integument used to distinguish P. xylostella from P. antiphona in New Zealand, and that the wing venation characters were variable in Australian populations, suggesting the presence of variants in Australia at the time. Interestingly, there were no significant differences in morphology between Australian and New Zealand P. xylostella except a slight difference in the sternite 7 length (longer in Australian P. xylostella). This may indicate that a single widespread invasion occurred to both countries (see Juric et al. (2017)).

Measurements of morphological features between adults caught in light traps and adults reared from field collected larvae and reared under laboratory conditions, exhibited morphological differences in both females and males. These findings suggest

that some morphological features vary depending on rearing conditions. Similarly, progeny of adults of one crop may differ from the adults from different crops. These findings suggest that adults collected from the field are suitable for taxonomic studies. The current study provides the first comprehensive, comparative analysis of genitalia measurements in P. australiana and P. xylostella. Overall, the findings indicate that most morphological characters examined here have limited capacity to differentiate both taxa in the absence of molecular data.

4.3 Summary and conclusion on the taxonomy of P. xylostella and P. australiana

Like Landry and Hebert (2013), the current study found that the CO1 barcode can be used to identify P. australiana. and P. xylostella. However, analysis of morphological features in the current study were not reliable in identifying the two taxa, except for the two morphological diagnostic features in females. These two features were consistent with the CO1 data and can therefore be used to identify female P. australiana from P. xylostella in future studies. This study has shown that three male characteristics (appearance of the vinculum saccus, valva structure at the ventro-distal margin and the ventral margin) and all male characteristics tested in this study and used by Robinson and Sattler (2001) and Landry and Hebert (2013) for species identification were not diagnostic. The ventro-distal margins described in Dugdale 1973 requires further examination.

Plutella xylostella taxonomy has been largely based on morphological characteristics such as genitalia features, wing venation, larval characteristics, pupal characteristics and adult external morphology of male and females (Baraniak, 2007; Clarke, 1971; Dugdale, 1973; Moriuti, 1986) and one study that examined the measurements of external morphology of Plutella populations in India (Chacko & Narayanasamy, 2004). However, there is evidence that sole reliance on morphological characters may be misleading, as has been found in the current study. For example, environmental conditions (such as temperature, humidity, light conditions and diets) can affect morphological development and lead to variation in morphological characters. Several studies have confirmed that diet influences the developmental and reproductive parameters in Plutella (Begum et al., 1996; Muhamad et al., 1994; Sarfraz, Dosdall, & Keddie, 2010; Shelton & Nault, 2004; Talekar & Shelton, 1993; Yamada, 1983). In

the current study, it has been shown that laboratory conditions have impacted the development of larvae leading to morphological differences in both females and males. Therefore, it is necessary that an integrated morphological and molecular framework is used to re-evaluate and resolve the systematic relationships of Plutella species worldwide.

4.4 Distribution, host range and pest status of the two taxa

4.4.1 Distribution This study has shown that P. australiana occurs sympatrically with P. xylostella in most locations in Queensland, New South Wales and Tasmania. While the presence of P. australiana in QLD and NSW further confirms the findings of Landry and Hebert (2013), the presence of P. australiana in Tasmania has been confirmed in this study. In the current study, P. australiana was not found to occur in New Zealand but a more extensive examination is required because of a number of records which suggest its possible presence. For example, Dugdale (1973) observed a curved tubular projection in a single female specimen and three female specimens collected from Ivercargill, Boulder Bank, Nelson in New Zealand in 1907 and one from Fiordland collected before 1910 were observed to have morphological characteristics of P. australiana. This was based on the S7 oval sclerotisation with no raised pair of folds and the antrum projection in the side view (curvature and tubular projection projecting for less than 1/3 its length beyond S7) (personal communication from John Dugdale, Senior Entomologist, New Zealand). However, specimens used in this study collected from New Zealand between 2008 and 2016 were entirely P. xylostella. As this study has shown, several of the morphological features identified by Landry and Hebert (2013) cannot be used to differentiate the two taxa, but the curvature of the female tubular projection is a consistent diagnostic feature, supporting the suggestion that P. australiana may also be present in New Zealand (Dugdale, 1973). These findings suggest that further examination of New Zealand populations should be conducted to identify diversity within Plutella spp. Similarly, investigation of Australian specimens from the Australian National Insect Collection (ANIC) may provide a good indication of any early presence of P. australiana in Australia.

The origins and distribution of P. australiana in the wider region are not known. Plutella australiana has not been found in New Caledonia (2015) (personal communication from Christian Mille, researcher in entomology, New Caledonia) and further examination on other islands is required. All current available CO1 barcode sequence data (NCBI, BOLD) for P. australiana are from Australian specimens. Further studies on the regional distribution and relationships to other Plutellidae are required.

4.4.2 Host preference and pest status of P. australiana

In the current study, host plants of P. xylostella were found to be cabbage, kale, broccoli, swedes, cauliflower and the weed field mustard (Brassica rapa). Moreover, they are known to damage canola crops and are considered to be a significant and sporadic pest in canola (Furlong et al., 2008), recolonising winter canola crops in southern and western regions of Australia (Perry et al., 2015).

This study is the first to describe the occurrence of P. australiana larvae on cabbage (Brassica oleracea) and field mustard (B. rapa). Plutella australiana is known to occur on canola, the cultivar of B. rapa, in South Australia (personal communication from Michael Keller, Professor, Adelaide). Three P. australiana larvae were previously found on Lincoln weed (Diplotaxis tenufolia, family Brassicaceae) in South Australia (Perry et al., 2015; Perry et al., 2017). The occurrence of P. australiana larvae on weedy field mustard in a kale field in Werombi (NSW) but not on kale itself, and the frequent co-occurrence of both taxa in light traps but only detection of P. xylostella larvae in the crop, indicate a host preference in P. australiana. Similarly, the detection of P. australiana on Lincoln weed but not on nearby canola crops suggests that P. australiana is not widely adapted to brassica crops (Perry et al., 2015). No P. australiana larvae were found on cauliflower and swedes in Hobart (TAS) from the same location where they were present in light traps, further supporting their host preference for weeds. However, weeds in Tasmania were not explored in this study and remain to be examined. Both taxa were found to co-occur on host plants found in this study (cabbage and field mustard) with similar results reported from South Australia where both taxa were found to co-occur on Lincoln weed (Perry et al., 2017).

Plutella xylostella are also known to use wild crucifer hosts between brassica cropping seasons (Harcourt, 1986; Talekar & Shelton, 1993; Yamada, 1983) but have clearly adapted to use brassica crops as a host. Host preference and oviposition preference in P. xylostella is known to be influenced by the nutrient value of host plants (Sarfraz, Dosdall, Blake, et al., 2010) and on the amount of glossy leaf wax in the host plant, which is a defense mechanism (Stoner, 1990). As an example, sulfur fertilizers and nitrogen are known to promote egg laying in P. xylostella (Badenes‐Perez et al., 2010; Furlong et al., 2013; Staley et al., 2010). Similarly, green leaf volatiles, isothiocyanates, nitriles, dimethyl trisulfide, and terpenes emitted by cabbage and canola (Girling et al., 2011; Kugimiya et al., 2010) may impact P. ausraliana host preference. However, this need further examination and may help to identify their target host plants.

Existence of P. xylostella on wild brassica crops as a refuge during the seasons which are not suitable for cultivation has been reported earlier (Furlong et al., 2013; Harcourt, 1986; Sarfraz et al., 2006; Talekar & Shelton, 1993). As a solution, Sarfraz et al. (2011) suggested that monitoring and controlling P. xylostella in weed species even before crops are cultivated may help control this pest in the fields, and may also be the case with P. australiana. Previous studies have reported the presence of DBM on different host plants (Begum et al., 1996; Löhr & Rossbach, 2001; Robinson & Sattler, 2001) so including host and native plants in Australia needs to be investigated to determine whether P. australiana is a native species.

Overall, these findings suggest that P. australiana is an occasional or emerging pest of brassica crops.

4.4.3 Summary and conclusions on the distribution, host range and pest status of the two taxa.

Overall, these findings suggest that P. australiana is an occasional or emerging pest of brassica cabbage crops. Preference of P. australiana for B. rapa suggests that canola crops widely grown in South Australia and Western Australia are at risk. Reports of P. australiana on canola, Lincoln weed and in light traps in South Australia already show a threat to the canola industry in Australia.

4.5 Pest management

Controlling P. xylostella through extensive usage of insecticides had led to a build-up of insecticide resistance to almost all the available insecticides (APRD, 2012; Ridland & Endersby, 2011; Sarfraz et al., 2005). All chemical insecticides have high mutagenic power and their extensive use can increase the number of mutations and create bottlenecks in DBM populations (Roux et al., 2007). For these reasons, integrated pest management strategies which include chemical, biological and cultural control tactics (Sarfraz et al., 2006) are best for effective control of P. xylostella. In particular, an emphasis on maintaining natural enemies has been advocated with common biological control agents including parasitoids, pathogenic fungi, bacteria and viruses. The existence of P. australiana in Australia has made these applications more challenging as their resistance to any of these is unknown. However, a recent study on the frequency of mutations associated with pyrethroid resistance in Australian populations showed that P. australiana were susceptible to insecticides (Perry et al., 2017).

As there is mounting evidence that P. australiana poses a possible risk to the Australian canola industry, it is critical that the status of P. australiana as a native or exotic species in Australia is clarified to avoid any delay in the release or registration of new biological controls. As an example, PlxyGV isolates which are being investigated as potential biological controls by QUT (Spence et al., 2016) will need to undergo extensive risk assessments on non-target organisms and host specificity prior to their release (BiosecurityAct, 2015).

4.6 Limitations and recommendations

This study adds to the existing knowledge of molecular, morphology, distribution and host plant use of Plutella species in Australia and New Zealand. However, there is still a significant knowledge gap regarding the life history of P. australiana and it presents issues for biological control, pest management and potentially, market access for Australian production.

A range of limitations were identified in this study. Firstly, to enable confirmation that the two taxa are in fact different species, fixed differences at nuclear DNA loci need

to be demonstrated. This was not possible in the current study due to time constraints. Secondly, while this study provides the most extensive examination of morphological characters in Plutella species undertaken to date, morphological identification used only the lateral view. This was due to the difficulty in examining the genitalia structures in their ventral position when handling large numbers of specimens. It is possible that characters which are diagnostic in identifying the two taxa may have been revealed if the ventral position had been examined. Finally, the number of male and female individuals used for morphological measurements varied because sex was not known when the initial collections were made. This lead to a biased sample number for males and females.

To expand the knowledge of P. australiana in Australia and other Plutella species in other countries, future research must use a combination of both mitochondrial and nuclear markers to examine the taxonomy of this species. Furthermore, morphological features should not be used in isolation to identify new species because environmental factors can influence morphology. A combined approach based on morphology and molecular data will decrease the potential for error. Additionally, adult specimens collected from the field are recommended for morphological examinations. Future studies should sample DBM from a wider geographical range across Australia (including WA and northern Australia). It is recommended that a broad range of both crop and weed varieties are examined to further clarify the host range of P. australiana. Such an investigation should also include a wide range of Australian native Brassicaceae plants. Further sampling should also be undertaken in New Zealand and other islands where it has been anecdotally recorded (example; Macquarie Island, Christian Mille pers comm). A wider systematic analysis across multiple locations is required to establish the origin of P. australiana.

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Appendices

Appendix A

Genbank accession numbers of the individuals taken for the phylogenetic analyses.

Accession number Species KF370582 P. xylostella KF370599 P. xylostella KF370638.1 P. xylostella KF370642 P. xylostella KF370657 P. xylostella KF370673 P. xylostella KF370770.1 P. xylostella KF370856 P. xylostella

KF370743 P. xylostella

KF370633 P. xylostella

KF370794 P. xylostella KF370724 P. xylostella KF370749 P. australiana KF370728 P. australiana KF370780 P. australiana KF370791 P. australiana KF370824 P. australiana KF370833 P. australiana KF370844 P. australiana KF370849 P. australiana KF370853 P. australiana KF370854 P. australiana KF370862 P. australiana KF370868 P. australiana HQ923231.1 Hyperxena scierana KF808771.1 Plutella hyperboreella KT140166.1 Plutella porrectella

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Appendix B

Summary of female genitalia morphological measurement data of adults collected from light traps taken for the analyses.

Year of Location Sample ID Molecular (CO1) Female measurements (μm) collection Upper Tubular Whole Whole Sternite 7 part projection length width Length length length (WL) (WW) (S7L) (UPL) (TPL) 2015 Werombi, NSW SYDW15_3 P. australiana 840.5 503.7 489.6 155.0 350.9 SYDW15_8 P. australiana 934.3 564.9 434.5 193.8 499.8 SYDW15_13 P. australiana 712.0 285.6 489.5 206.0 222.5 2015 Mowbray Park, 2015 M15_2_Nov P. australiana 837.8 469.2 390.3 158.1 447.6 M15_3_Nov P. australiana 829.6 520.2 338.3 176.8 491.3 M15_4_Nov P. australiana 736.1 462.4 253.3 195.5 482.8 M15_5_Nov P. australiana 758.2 455.6 343.3 212.5 414.9 M15_6_Nov P. australiana 719.1 464.1 307.7 161.5 411.4 M15_8_Nov P. australiana 654.5 464.1 334.9 180.2 319.6 M15_9_Nov P. australiana 695.3 448.8 334.9 197.2 360.4 M15_10_Nov P. australiana 717.4 498.1 316.2 173.4 401.2 M15_12_Nov P. australiana 661.3 445.4 289.0 170.0 372.3 M15_13_Nov P. australiana 770.1 508.3 372.3 156.4 397.8 M15_11_Nov P. xylostella 642.6 408.0 236.3 171.7 406.3

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Appendix B Continued

2014 Birkdale, QLD BIR1 P. xylostella 668.1 496.4 273.7 231.2 394.4 2014 Hobart, QLD HOB3_12 P. xylostella 749.7 445.4 368.9 236.3 380.8 HOB3_13 P. xylostella 683.4 436.9 187.0 209.1 496.4 HOB3_14 P. xylostella 705.5 554.2 263.5 181.9 442.0 HOB3_18 P. xylostella 751.4 465.8 360.4 202.3 391.0 2014 Theresa Park, NSW SYDT1 P. xylostella 867.9 445.0 382.0 210.1 485.9 SYDT4 P. xylostella 799.6 368.6 376.6 188.3 423.0 SYDT6 P. xylostella 646.8 406.8 177.4 202.0 469.4 SYDT8 P. xylostella 968.8 513.3 431.2 245.6 537.6 SYDT11 P. xylostella 780.5 466.9 390.0 199.2 390.5 SYDT20 P. xylostella 674.9 481.0 236.3 251.6 438.6 2014 Werombi, NSW SYDW1 P. xylostella 734.1 431.4 215.6 188.3 518.5 SYDW2 P. xylostella 679.6 415.0 354.8 212.9 324.8 SYDW3 P. australiana 597.7 469.6 223.8 141.9 373.9 SYDW4 P. xylostella 780.5 466.9 311.1 232.0 469.4 SYDW5 P. xylostella 742.3 488.7 204.7 229.2 537.6 SYDW6 P. xylostella 698.7 447.7 155.6 234.7 543.1 SYDW7 P. xylostella 581.5 NA NA 199.2 NA SYDW8 P. xylostella 698.7 538.0 199.2 240.2 499.4 SYDW9 P. xylostella NA 488.7 NA 191.0 NA SYDW10 P. xylostella 780.5 431.4 267.5 199.2 513.1

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Appendix B Continued

2015 Werombi, NSW SYDW15_6 P. xylostella NA 471.1 NA 151.0 NA SYDW15_7 P. xylostella 854.8 540.4 381.5 220.3 473.3 SYDW15_9 P. xylostella 899.6 526.2 397.8 189.7 501.8 SYDW15_10 P. xylostella 956.8 505.8 438.6 NA 518.2 SYDW15_11 P. xylostella 818.0 328.4 322.3 236.6 495.7 SYDW15_12 P. xylostella 836.4 520.0 359.0 210.1 477.4 2016 New Zealand DBM1_5 P. xylostella 836.4 485.4 385.6 244.8 450.8 DBM1_6 P. xylostella 838.4 526.2 363.1 222.4 475.3 DBM1_9 P. xylostella 767.0 499.6 357.0 236.6 410.0 DBM1_11 P. xylostella 844.6 489.5 375.4 238.7 469.2 DBM1_12 P. xylostella 799.6 573.8 425.7 259.3 373.9 2013 New Zealand DBM4_1 P. xylostella 664.7 404.6 300.9 207.4 363.8 DBM4_6 P. xylostella 776.9 440.3 370.6 232.9 406.3 DBM4_11 P. xylostella 674.9 440.3 256.7 175.1 418.2 DBM4_17 P. xylostella 654.5 404.6 229.5 209.1 425.0 2013 New Zealand DBM5_11 P. xylostella 809.2 467.5 346.8 258.4 462.4 DBM5_16 P. xylostella 705.5 469.2 311.1 219.3 394.4 DBM5_17 P. xylostella 639.2 447.1 166.6 197.2 472.6 DBM5_21 P. xylostella 834.7 493.0 396.1 243.1 438.6 2008 New Zealand DBM9_1 P. xylostella 727.6 460.7 294.1 207.4 433.5 DBM9_3 P. xylostella 722.5 486.2 282.2 222.7 440.3 DBM9_4 P. xylostella 617.1 464.1 170.0 214.2 447.1 DBM9_5 P. xylostella 669.8 404.6 312.8 226.1 357.0

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Appendix B Continued

DBM9_7 P. xylostella 639.2 452.2 193.8 173.4 445.4 DBM10_1 P. xylostella 822.8 472.6 470.9 212.5 351.9 DBM10_2 P. xylostella 710.6 430.1 283.9 178.5 426.7 DBM10_3 P. xylostella 674.9 474.3 272.0 210.8 402.9 DBM10_4 P. xylostella 765.0 413.1 375.7 192.1 389.3 DBM10_5 P. xylostella 678.3 481.1 272.0 205.7 406.3 DBM10_6 P. xylostella 790.5 467.5 374.0 234.6 416.5 DBM10_12 P. xylostella 816.0 477.7 448.8 249.9 367.2

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Appendix C

Summary of male genitalia morphological measurement data of adults collected from light traps taken for the analyses.

Year of Molecular Location Sample ID Male measurements (μm) collection (CO1) Vinculum Whole Valva Valva Phallus saccus length width length length length (WL) (VW) (VL) (PL) (VSL) 2014 Birkdale, QLD BIR2 P. australiana 1074.4 370.6 656.2 598.4 418.2 BIR4 P. australiana 1064.2 328.1 598.4 NA 465.8 BIR3 P. xylostella 989.4 294.1 625.6 503.2 363.8 BIR5 P. xylostella 1008.1 299.2 632.4 510 375.7 2014 Hobart, QLD HOB3_6 P. australiana 999.6 340 623.9 578 375.7 HOB3_10 P. australiana 972.4 329.8 584.8 540.6 387.6 HOB3_17 P. australiana NA 290.7 586.5 NA NA HOB3_2 P. xylostella 980.9 280.5 678.3 484.5 302.6 HOB3_3 P. xylostella 972.4 312.8 591.6 562.7 380.8 HOB3_4 P. xylostella NA 300 588.2 489.6 NA HOB3_5 P. xylostella NA 306 591.6 494.7 NA 2015 Theresa Park, NSW SYD15T_3_Oct P. australiana 982.6 341.7 635.8 593.3 346.8 SYD15T_21_Oct P. australiana 1020 316.2 642.6 617.1 377.4 SYD15T_22m_Oct P. australiana 1037 300.9 598.4 521.9 438.6 SYD15T_29_Oct P. australiana 952 292.4 571.2 532.1 380.8

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Appendix C Continued

SYD15T_16m_Oct P. australiana 972.4 302.6 603.5 559.3 368.9 SYD15T_17m_Oct P. australiana 1004.7 338.3 608.6 557.6 396.1 SYD15T_30_Oct P. australiana 994.5 306 584.8 561 409.7 SYD15T_18m_Oct P. xylostella 972.4 324.7 598.4 494.7 374 SYD15T_19m_Oct P. xylostella 999.6 323 615.4 482.8 384.2 SYD15T_20m_Oct P. xylostella 1018.3 299.2 608.6 532.1 409.7 2014 Theresa Park, NSW SYDT17 P. xylostella 899.3 309.4 615.4 476 283.9 2014 Samford, QLD SAM1 P.xylostella 1033.6 360.4 612 515.1 421.6 SAM7 P.xylostella 986 273.7 652.8 428.4 333.2 SAM9 P.xylostella 1025.1 312.8 606.9 499.8 418.2 2016 New Zealand DBM1_1 P. xylostella 948.6 302.6 600.1 452.2 348.5 DBM1_2 P. xylostella 1054.35 312.12 632.2 493 422.15 DBM1_3 P. xylostella 1008.1 326.4 596.7 521.9 411.4 DBM1_4 P. xylostella 1004.7 333.2 627.3 489.6 377.4 DBM1_7 P. xylostella 1047.2 324.7 668.1 511.7 379.1 2016 New Zealand DBM3_1 P. xylostella 1030.2 368.9 666.4 518.5 363.8 DBM3_2 P. xylostella 989.4 326.4 613.7 511.7 375.7 DBM3_3 P. xylostella 1031.9 319.6 656.2 501.5 375.7 DBM3_4 P. xylostella 1048.9 312.8 661.3 496.4 387.6 DBM3_5 P. xylostella 962.2 309.4 603.5 479.4 358.7 DBM3_6 P. xylostella 1025.1 326.4 688.5 513.4 336.6 DBM3_7 P. xylostella 965.6 324.7 629 476 336.6 2013 New Zealand DBM4_3 P. xylostella 1052.3 321.3 632.4 503.2 419.9

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Appendix C Continued

DBM4_4 P. xylostella 970.7 324.7 634.1 501.5 336.6 DBM4_5 P. xylostella 991.1 345.1 622.2 NA 368.9 DBM4_8 P. xylostella 906.1 297.5 552.5 487.9 353.6 DBM4_9 P. xylostella 955.4 346.8 586.5 498.1 368.9 2013 New Zealand DBM5_12 P. xylostella 977.5 345.1 630.7 520.2 346.8 DBM5_15 P. xylostella 936.7 299.2 601.8 499.8 334.9 DBM5_18 P. xylostella 967.3 278.8 555.9 527 411.4 DBM5_19 P. xylostella 974.1 326.4 608.6 510 365.5 DBM5_22 P. xylostella 941.8 312.8 612 484.5 329.8 DBM5_26 P. xylostella 963.9 316.2 617.1 498.1 346.8 2008 New Zealand DBM9_2 P. xylostella 882.3 292.4 562.7 443.7 319.6 DBM9_6 P. xylostella 863.6 300.9 544 481.1 319.6 DBM9_8 P. xylostella 938.4 268.6 562.7 457.3 375.7 DBM9_10 P. xylostella 841.5 256.7 535.5 450.5 306 DBM9_13 P. xylostella 975.8 278.8 613.7 491.3 362.1 DBM10_19 P. xylostella 975.8 299.2 605.2 508.3 370.6

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Appendix D

Bipartition maximum likelihood (ML) tree with bootstrap values. The tree was collapsed to remove low supported nodes (≥75%). For the full tree view go to: https://1drv.ms/b/s!AjKrDFNSXOJphq1UQfDEnH0kiVYWgw

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Appendix E

Bayesian analysis with posterior probabilities. The tree was collapsed to remove low supported nodes (≥75%). For the full tree view go to: https://1drv.ms/b/s!AjKrDFNSXOJphroJo2cRme38DE6gug

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Appendix F

Male genitalia specimens showing sinuation in the ventral margin of the valva in (A) P. xylostella (specimen HOB4_1) and no sinuation in (B) P. australiana (specimen TL15_4) which contradict the description in Landry and Hebert (2013).

A) B)

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Appendix G

Summary of female genitalia morphological measurement data of adults reared from field collected larvae taken for the analyses.

Year of Molecular Location Crop type Sample ID Female measurements (μm) collection (CO1) Upper Tubular Whole Whole Sternite part projection length width 7 Length length length (WL) (WW) (S7L) (UPL) (TPL) 2015 Theresa Park, NSW cabbage TL15_3 P. australiana 768.4 464.1 442.0 173.4 326.4 TL15_6 P. australiana 739.5 433.5 326.4 144.5 413.1 TL15_7 P. australiana 739.5 457.3 408.0 180.2 331.5 TL15_8 P. australiana 734.4 436.9 392.7 144.5 341.7 TL15_9 P. australiana 732.7 435.2 377.4 154.7 355.3 TL15_10 P. australiana 765.0 459.0 351.9 176.9 413.1 TL15_12 P. xylostella 661.3 375.7 367.2 147.9 294.1 TL15_19 P. xylostella 656.2 431.8 368.9 202.3 287.3 TL15_11 P. australiana 736.1 484.5 379.1 178.5 357.0 2015 Werombi, NSW weed (field mustard) WW15_3 P. australiana 727.6 421.6 416.5 147.9 311.1 WW15_4 P. australiana 751.4 453.9 402.9 147.9 348.5 WW15_6 P. australiana 659.6 455.6 306.0 158.1 353.6 WW15_8 P. australiana 812.6 442.0 445.4 158.1 367.2 WW15_2 P. australiana 759.9 455.6 363.8 149.6 396.1

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Appendix G Continued

WW15_9 P. australiana 787.1 431.8 397.8 161.5 389.3 2014 Hobart, TAS cauliflower, swedes HOB4_3 P. xylostella 829.6 453.9 392.7 107.2 436.9 HOB4_4 P. xylostella 803.8 495.6 316.2 213.4 487.6 HOB4_5 P. xylostella 792.2 498.1 285.6 200.6 506.6 HOB4_6 P. xylostella 840.5 471.1 348.8 193.8 491.6 HOB4_7 P. xylostella 854.8 571.0 320.3 193.8 534.5 HOB4_8 P. xylostella 830.3 505.8 471.2 206.0 359.0 HOB4_9 P. xylostella 789.5 475.2 438.6 212.2 350.9 HOB4_10 P. xylostella 852.7 460.9 438.6 175.4 414.1 2015 Werombi, NSW kale WL15_6 P. xylostella 775.2 508.3 275.4 268.6 499.8 WL15_7 P. xylostella 963.4 546.1 423.0 272.9 540.4 WL15_8 P. xylostella 831.3 511.7 430.1 244.8 401.2 WL15_9 P. xylostella 922.5 500.9 510.0 226.7 412.5 WL15_10 P. xylostella 816.0 477.7 363.8 204.0 452.2

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Appendix H

Summary of male genitalia morphology measurement data of adults reared from field collected larvae taken for the analyses.

Year of Molecular Location Crop type Sample ID Male measurements (μm) collection (CO1) Vinculum Whole Valva Valva Phallus saccus length width length length length (WL) (VW) (VL) (PL) (VSL) 2015 Theresa Park, NSW cabbage TL15_4 P. australiana 935 290.7 557.6 545.7 377.4 TL15_5 P. australiana 940.1 331.5 527 533.8 413.1 TL15_13 P. australiana 984.3 304.3 593.3 543.7 391 TL15_14 P. australiana 960.5 317.9 552.5 NA 408 2014 Hobart, TAS cauliflower, swedes HOB4_1 P. xylostella 980.9 312.8 559.3 472.6 421.6 HOB4_2 P. xylostella 1033.6 317.9 639.2 481.1 394.4 HOB4_11 P. xylostella 1004.7 300.9 642.6 489.6 362.1 HOB4_12 P. xylostella 979.2 324.7 646 499.8 333.2 HOB4_13 P. xylostella 936.7 328.1 574.6 470.9 362.1 TL15_28 P. xylostella 846.6 307.7 525.3 450.5 321.3 2015 Werombi, NSW kale WL15_21 P. xylostella 938.4 309.4 705.5 460.7 232.9 WL15_22 P. xylostella 999.6 312.8 693.6 477.7 306 WL15_23 P. xylostella 952 299.2 627.3 482.8 324.7 WL15_24 P. xylostella 1011.5 294.1 598.4 479.4 413.1 WL15_25 P. xylostella 963.9 329.8 559.3 472.6 404.6

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Appendix H Continued

2015 Werombi, NSW weed (field mustard) WW15_16 P. australiana 895.9 317.9 566.1 562.7 329.8 WW15_18 P. xylostella 890.8 261.8 503.2 516.8 387.6 WW15_17 P. australiana 1011.5 333.2 608.6 567.8 402.9 WW15_19 P. australiana 991.1 317.9 579.7 559.3 411.4

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Supplementary Materials

Conference: Australian Entomological Society annual conference, 2015, Cairns, Queensland.

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Conference: International Congress of Entomology, 2016, Orlando, Florida.

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Grants received

1) Australian Government’s National Taxonomy Student Travel Grant 2014-15 of the Research Grant Programme (NTRGP) - Australian Biological Resources Study (ABRS) – The amount of AUD 825 was used to travel to the AES conference and the registration of the same conference.

Membership of professional societies 1. Australian Entomological Society. 2. Entomological society of America.

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