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Systematics, Biology, and Behavior of Fruit-Piercing and Blood-Feeding Moths in the Subfamily Calpinae (Lepidoptera

Permanent Link: http://ufdc.ufl.edu/UFE0023641/00001

Material Information

Title: Systematics, Biology, and Behavior of Fruit-Piercing and Blood-Feeding Moths in the Subfamily Calpinae (Lepidoptera Noctuidae)
Physical Description: 1 online resource (239 p.)
Language: english
Creator: Zaspel, Jennifer
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: blood, endosymbiont, evolution, fruit, morphology
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: A phylogenetic review of the fruit-piercing and blood-feeding tribe Calpini Lepidoptera: Noctuidae: Calpinae was conducted to determine the evolutionary relationships among the genera. The evolution of feeding behaviors was investigated by using the resulting phylogeny. A set of morphological characters was compiled and included characters from the head, appendages, male and female genitalia. Structures associated with the proboscis were examined in these moths using both light microscopy and SEM methods. A morphological data matrix with 66 characters was compiled for 65 taxa. A reduced molecular data set includes nearly complete sequences of the cytochrome oxidase I mitochondrial gene and a fragment of the nuclear large subunit 28S rRNA for 34 taxa. Phylogenetic trees of separate and combined data sets were constructed using parsimony and Bayesian analyses. Binary feeding behavior characters were coded for all taxa in the morphological matrix and mapped onto the resulting topology using parsimony optimizations. Results from the analysis based on morphological data suggest Calpini is monophyletic and is supported by five synapomorphies. Three of these are unreversed, shared characters of the proboscis. Tearing hooks are restricted to the Calpini and little additional variation within the tribe exists suggesting proboscis morphology may not be strongly correlated with feeding behavior. The results from this study support the hypothesis that hematophagy in the genus Calyptra evolved from the fruit-piercing habit as opposed to tear feeding. A preliminary checklist of Calpini is also provided, incorporating corrections and changes to publication dates and nomenclature as presented in recent checklists. A microbial survey of Calyptra thalictri Borkhausen using polymerase chain reaction PCR primers for 16S rRNA sequences for Eubacteria. High-fidelity PCR and subsequent DNA analyses indicated that at least five microorganisms belonging to the ?, ?, and ? Proteobacteria were present. Two eubacterial sequences, related to a Klebsiella sp. and a Sinorhizobium sp., were detected in the abdomens individuals sampled, and three additional sequences were found in some samples, suggesting all five could be associated with abdominal structures. No Archaea, Fungi including yeast-like organisms, Microsporidia, or Wolbachia were detected. These results document the first microbial associates in a fruit-piercing and blood-feeding moth.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jennifer Zaspel.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Branham, Marc A.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0023641:00001

Permanent Link: http://ufdc.ufl.edu/UFE0023641/00001

Material Information

Title: Systematics, Biology, and Behavior of Fruit-Piercing and Blood-Feeding Moths in the Subfamily Calpinae (Lepidoptera Noctuidae)
Physical Description: 1 online resource (239 p.)
Language: english
Creator: Zaspel, Jennifer
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: blood, endosymbiont, evolution, fruit, morphology
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: A phylogenetic review of the fruit-piercing and blood-feeding tribe Calpini Lepidoptera: Noctuidae: Calpinae was conducted to determine the evolutionary relationships among the genera. The evolution of feeding behaviors was investigated by using the resulting phylogeny. A set of morphological characters was compiled and included characters from the head, appendages, male and female genitalia. Structures associated with the proboscis were examined in these moths using both light microscopy and SEM methods. A morphological data matrix with 66 characters was compiled for 65 taxa. A reduced molecular data set includes nearly complete sequences of the cytochrome oxidase I mitochondrial gene and a fragment of the nuclear large subunit 28S rRNA for 34 taxa. Phylogenetic trees of separate and combined data sets were constructed using parsimony and Bayesian analyses. Binary feeding behavior characters were coded for all taxa in the morphological matrix and mapped onto the resulting topology using parsimony optimizations. Results from the analysis based on morphological data suggest Calpini is monophyletic and is supported by five synapomorphies. Three of these are unreversed, shared characters of the proboscis. Tearing hooks are restricted to the Calpini and little additional variation within the tribe exists suggesting proboscis morphology may not be strongly correlated with feeding behavior. The results from this study support the hypothesis that hematophagy in the genus Calyptra evolved from the fruit-piercing habit as opposed to tear feeding. A preliminary checklist of Calpini is also provided, incorporating corrections and changes to publication dates and nomenclature as presented in recent checklists. A microbial survey of Calyptra thalictri Borkhausen using polymerase chain reaction PCR primers for 16S rRNA sequences for Eubacteria. High-fidelity PCR and subsequent DNA analyses indicated that at least five microorganisms belonging to the ?, ?, and ? Proteobacteria were present. Two eubacterial sequences, related to a Klebsiella sp. and a Sinorhizobium sp., were detected in the abdomens individuals sampled, and three additional sequences were found in some samples, suggesting all five could be associated with abdominal structures. No Archaea, Fungi including yeast-like organisms, Microsporidia, or Wolbachia were detected. These results document the first microbial associates in a fruit-piercing and blood-feeding moth.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jennifer Zaspel.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Branham, Marc A.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0023641:00001


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SYSTEMATICS, BIOLOGY, AND BEHAVI OR OF FRUIT-PIERCING AND BLOODFEEDING MOTHS IN THE SUBFAMILY CA LPINAE (LEPIDOPTERA: NOCTUIDAE) By JENNIFER MICHELLE ZASPEL A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008 1

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2008 Jennifer M. Zaspel 2

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To Dr. Hans Bnziger for assistance with this project and for his di scovery of blood-feeding moths in the genus Calyptra 3

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ACKNOWLEDGMENTS First and foremost, I thank my advisor and chair of my graduate committee, Dr. Marc A. Branham and the members of my graduate co mmittee, Dr. Marjorie A. Hoy, Dr. Jacqueline Miller, and Dr. David Reed for their professiona l advice, scientific guidance, and financial support. I also thank Dr. Hans Bnziger and Michael Fibiger for many helpful discussions about Calyptra I would like to thank Drs. A. Jeyaprakash and J. Meyer for their technical advice and laboratory training in molecular biology. Vladimiar S. Kononenko was instrumental in organizing the expediti ons to far eastern Russia and for the acquisition of the specimens used in several studies in my dissertation. I would also like to thank my fi eld guide on both expeditions in Russia, Boris Popkov, the staff of the Hunting Area, and the resear ch scientists at Gornotayeznaya Biological Station. I also greatly appreciate the assistance of Ms. Valen tina Kolesnikova from the Russian Academy of Sciences Far Eastern Branch for her assistance in obtaining permits for collecting. I also thank Susan Weller and Harald Krenn for suggestions on the comparative mouthpart survey of calpine noctuids (Chapter 2); Hans Bnziger, Roland Hi lgartner, and Harry Fay kindly provided adult feeding images figured in the chapter. Drs. R. Rougerie, M. Hajibabaei, D. Janzen, W. Hallwachs, and P. Hebert assistan ced in obtaining barcode sequences for some taxa used in the molecular phylogenetic study. I also acknowledge all individual s and institutions listed in Chapter 3 for their assistance in obtaining specimens for the morphological and molecular phylogenetic study. I gratefully acknowledge the Fl orida Department of Ag riculture, Division of Plant Industries, Gainesville, FL for the use of their SEM and Dr. Paul Skelly for his assistance with the imaging equipment as well as Bra nden Apitz for assistance with SEM and figure formatting. The following people are acknowledged for their assistance in data collection for chapter 7: M.E. Sharf and his graduate students for the use of the QT-RT PCR machine in his 4

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laboratory, Paul Shirk for supplying specimens, and D.G. Boucias for the use of his spectrophotometer. Finally, I w ould like to thank past and present members of Branham, Hoy, McGuire Center, Reed, and Welle r laboratories over the years for their support and numerous scientific discussions. This work would not have been possible w ithout the support of my family and their encouragement throughout my graduate studies This work was supported in part by the American Philosophical Society, Davies, Fischer, and Eckes Endowment in Biological Control to Marjorie A. Hoy at the University of Florida, Explorers Club, Florida Entomological Society, National Geographic Societys Fund for Resear ch and Exploration, National Park Service Inventories and Monitoring Program, National Science Foundation (DDIG, DEB-0807975), Systematics Research Fund, and the Vam York Scholarship Fund. 5

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES ...........................................................................................................................9 LIST OF FIGURES .......................................................................................................................11 ABSTRACT ...................................................................................................................................13 CHAPTER 1 INTRODUCTION................................................................................................................. .15 Literature Review ...................................................................................................................15 Systematics of Calpini .....................................................................................................18 Overview of Fruit-Piercing and Bl ood-Feeding Moths in Tribe Calpini ........................15 Proboscis morphlogy in piercing moths ..........................................................................20 Blood feeding and endosymbionts in Calpini .................................................................21 Research Objectives ................................................................................................................24 2 A COMPARATIVE SURVEY OF PROBOSCIS MORPHOLOGY AND ASSOCIATED STRUCTURES IN FR UIT-PIERCING AND BLOOD-FEEDING MOTHS IN THE SUBFAMILY CALPI NAE (LEPIDOPTERA: NOCTUIDAE)...............31 Introduction .............................................................................................................................31 Terminology ....................................................................................................................34 Materials and Methods ...........................................................................................................34 Characterization of Functional Feeding Catergories .......................................................35 Specimen Preparation ......................................................................................................39 Results .....................................................................................................................................41 Description of the piercing structures of the probosci s visible by light microscopy ......41 Description of the structures vi sible by scanning electron microscopy ..........................43 Discussion ...............................................................................................................................51 3 RECONSTRUCTING THE EVOLUT IONARY RELATIONSHPS OF THE VAMPIRE MOTHS AND THEIR FRUI T-PIERCING RELATIVES USING MORPHOLOGICAL AND MOLECULAR DATA (LEPIDOPTERA: NOCTUIDAE: CALPINAE: CALPINI) .........................................................................................................74 Introduction .............................................................................................................................74 Taxon Sampling ...............................................................................................................76 Materials and Methods ...........................................................................................................76 Morphological Data .........................................................................................................78 Molecular Data ................................................................................................................80 6

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Phylogenetic Analyses .....................................................................................................81 Results and Discussion ...........................................................................................................83 Evolution of Feeding Behavior s and Complementary Analyses .....................................82 Summary of Morphological Character Variation ............................................................83 Phylogenetic Analysis of Morphological Data ................................................................88 Molecular Data and Combined Analyses ........................................................................89 Evolution of Feeding Behavior s and Complementary Analysis .....................................91 Conclusions .............................................................................................................................92 4 WORLD CHECKLIST OF TRIBE CALPINI (LEPIDOPTERA: NOCTUIDAE: CALPINAE).........................................................................................................................125 Introduction ...........................................................................................................................125 Calpini ...........................................................................................................................127 Checklist ...............................................................................................................................127 Genus and tribal placement undetermined ....................................................................140 5 ANOTHER BLOOD FEEDER? EXPERI MENTAL FEEDING OF A FRUITPIERCING MOTH SPECIES ON HU MAN BLOOD IN THE PRIMORYE TERRITORY OF FAR EASTERN RUSSIA (LEPIDOPTERA: NOCTUIDAE: CALPINAE).........................................................................................................................141 Introduction ...........................................................................................................................141 Description of observation sites ....................................................................................142 Materials and Methods .........................................................................................................142 Experimental methods ...................................................................................................144 Results ...................................................................................................................................145 Discussion .............................................................................................................................148 5 MICROBIAL DIVERSITY ASSOCIATED WITH THE FRUI T-PIERCING AND BLOOD-FEEDING MOTH Calyptra thalictri (LEPIDOPTERA: NOCTUIDAE: CALPINAE) .........................................................................................................................164 Introduction ...........................................................................................................................164 Specimens ......................................................................................................................165 Materials and Methods .........................................................................................................165 Surface Sterilization ......................................................................................................166 DNA Extraction .............................................................................................................167 High Fidelity Polymerase Chain Reaction ....................................................................168 Cloning and Restriction Fragment Length Polymorphism Analysis .............................168 Results and Discussion .........................................................................................................169 High-fidelity PCR Amplification of Microbial Associates in C. thalictri ....................169 Microbial Associates of C. thalictri ...............................................................................170 7

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7 COMPARISON OF SHORT-TERM PRESERVATION AND ASSAY METHODS FOR THE MOLECULAR DETECTION OF WOLBACHIA IN THE MEDITERRANEAN FLOUR MOTH EPHESTIA KUEHNIELLA .....................................177 Scientific Note ......................................................................................................................177 Results ...................................................................................................................................181 Materials and Methods .........................................................................................................178 Discussion .............................................................................................................................182 8 PERSPECTIVES................................................................................................................. .186 APPENDIX A DATA MATRIX USED TO PRODUCED TREES BASED ON MORPHOLOGICAL DATA...................................................................................................................................191 B DATA MATRIX USED TO PRODUCE TREES BASED ON MORPHOLOGICAL AND MOLECULAR DATA................................................................................................194 LIST OF REFERENCES .............................................................................................................214 BIOGRAPHICAL SKETCH .......................................................................................................239 8

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LIST OF TABLES Table page 2-1 Sensilla and other structur es associated with calpine proboscides and their proposed function ..............................................................................................................................54 2-2 Specimens examined A. ....................................................................................................55 3-1 Specimens examined B. .....................................................................................................95 3-2 PCR conditions and sequences of primers used. .............................................................100 3-3 Known feeding behavior reports for Calpini and related genera included in complementary analyses ..................................................................................................101 3-4 Character support for major clades. .................................................................................104 5-1 Summary of feeding behaviors for moth specimens collected in Primorye Terriotry of Far Eastern Russia from July 14t-15th 2006 and July 17-20th 2006............................151 6-1 Original and phylotype-specific forward and reverse primers designed to detect microbial sequences in DNA isolated from C. thalictri ..................................................175 6-2 Pairwise sequence divergences (uncorrect ed p) between eubacterial phylotypes from C. thalictri and closely related 16S rRNA sequences using 1410-1504 bp of sequences. ........................................................................................................................176 7-1 Summary of RTQ PCR and spectrophotometry data for E. kuehniella specimens stored for a 2-yr. period compared to fresh specimens. ...................................................184 2-1 Sensilla and other structur es associated with calpine proboscides and their proposed function ..............................................................................................................................54 2-2 Specimens examined A. ....................................................................................................55 3-1 Specimens examined B. .....................................................................................................95 3-2 PCR conditions and sequences of primers used. .............................................................100 3-3 Known feeding behavior reports for Calpini and related genera included in complementary analyses ..................................................................................................101 3-4 Character support for major clades. .................................................................................104 5-1 Summary of feeding behaviors for moth specimens collected in Primorye Terriotry of Far Eastern Russia from July 14t-15th 2006 and July 17-20th 2006............................151 9

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6-1 Original and phylotype-specific forward and reverse primers designed to detect microbial sequences in DNA isolated from C. thalictri ..................................................175 6-2 Pairwise sequence divergences (uncorrect ed p) between eubacterial phylotypes from C. thalictri and closely related 16S rRNA sequences using 1410-1504 bp of sequences. ........................................................................................................................176 7-1 Summary of RTQ PCR and spectrophotometry data for E. kuehniella specimens stored for a 2-yr. period compared to fresh specimens. ...................................................184 10

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LIST OF FIGURES Figure page 1-1 Calyptra thalictri male feeding on human thumb (JMZ) in Russia 2008. .........................30 2-1 Feeding behaviors of adult moths in the subfamily Calpinae. ...........................................58 2-2 Description of proboscis regions; Oraesia rectistria .........................................................59 2-3 Examples of proboscis structures visibl e by light microscopy in selected feeding categories. ..........................................................................................................................60 2-4 Proboscis of non-calpine classical tear feeder ...................................................................61 2-5 Proboscides of taxa in th e non-piercing fruit-sucking group .............................................62 2-6 Examples of proboscis structures visi ble by scanning light microscopy in primary piercers of thick-skinned fruit but se condary piercers of hard-skinned fruit. ....................63 2-7 Examples of proboscis structures visi ble by scanning light microscopy in primary piercers of thick-skinned fruit but se condary piercers of hard-skinned fruit II. ................66 2-8 Examples of proboscis structures visi ble by scanning light microscopy in primary piercers of thick-skinned fruit but se condary piercers of hard-skinned fruit III. ...............68 2-9 Proboscis of taxa in the primary piercing of hard-skinned fruits group. ...........................69 2-10 Proboscides of taxa in the mammalia n skin-piercing and blood-feeding group. ...............70 2-11 Proboscides of taxa in the mammalian skin-piercing and blood-feeding group II............71 2-12 Proboscides of taxa in the tear-drinking group. .................................................................72 2-13 Uncertain taxa. ...................................................................................................................73 3-1 Blood-feeding moth, Calyptra thalictri ...........................................................................106 3-2 Proposed hypotheses for th e evolution of tear feedi ng and blood feeding within Lepidoptera. .....................................................................................................................107 3-3 Shape of labial palp segment II, Character 17 .................................................................108 3-4 Shape of labial palp segment III, Character 18 ................................................................109 3-5 Shape of saccular process (entire), Character 22 .............................................................110 3-6 Shape of saccular process = SaP (branched), Sa = saccus, Character 23 ........................111 11

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3-8 Shape of saccus, Character 27 .........................................................................................113 3-9 Shape of dorsal tegumen, Character 37 ...........................................................................114 3-10 Shape of uncus base 38 ....................................................................................................115 3-11 Shape of the posterior edge of the antevaginal plate (segment VII) ................................116 3-12 Shape of the posterior edge of segment VIII, Character 46 .............................................117 3-13 Shape of cervical sclerites of the corp us bursa, Character 50 ..........................................118 3-14 Shape of the corpus bursa, Character 52 ..........................................................................119 3-15 Shape of appendix bursa (AB), Character 60 ..................................................................120 3-17 Evolution of adult feed ing behaviors in Calpini. .............................................................122 3-18 Preliminary strict consensus tree of si x most parsimonius rearrangements for 34 taxa based on combined data set..............................................................................................123 3-19 Preliminary Bayesian analysis resulting from a simultaneous analysis of all data partitions for 34 taxa. .......................................................................................................124 5-1 Adult habitus image. Calyptra thalictri Male. ...............................................................153 5-2 Adult habitus image. Calyptra lata Male. .....................................................................154 5-3 Adult habitus image. Calyptra hokkaida, Male. .............................................................155 5-4 Proboscis of Calyptra thalictri : TH = Tearing Hooks. ....................................................156 5-5 Map of Primorye Region of Far Eastern Russia. .............................................................157 5-6 Primary collecting site 1. ................................................................................................158 5-7 Primary collecting site 2. .................................................................................................159 5-8 Map of primary collecting site 1. ....................................................................................160 5-9 Map of primary collecting site 2. ....................................................................................161 5-10 Image of Calyptra thalictri feeding on human thumb (JMZ). .........................................162 5-10 Calyptra thalictri feeding on raspberry during night observations.................................163 7-1 Examination of DNA preservation and amplification by HF PCR of the wsp A gene fragment (605 bp) in E. kuehniella ..................................................................................185 12

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy SYSTEMATICS, BIOLOGY, AND BEHAVI OR OF FRUIT-PIERCING AND BLOOD-FEEDING MOTHS IN THE SUBF AMILY CALPINAE (LEPIDOPTERA: NOCTUIDAE) By Jennifer Michelle Zaspel December 2008 Chair: Marc A. Branham Major: Entomology and Nematology A phylogenetic review of the fruit-piercing a nd blood-feeding tribe Ca lpini [Lepidoptera: Noctuidae: Calpinae] was conducted to determ ine the evolutionary relationships among the genera. The evolution of feeding behaviors wa s investigated by using the resulting phylogeny. A set of morphological characte rs was compiled and included characters from the head, appendages, male and female genitalia. Structur es associated with the proboscis were examined in these moths using both light microscopy a nd SEM methods. A morphological data matrix with 66 characters was compiled for 65 taxa. A reduced molecular data set includes nearly complete sequences of the cytochrome oxidase I mitochondrial gene and a fragment of the nuclear large subunit 28S rRNA for 34 taxa. Phylogenetic trees of separate and combined data sets were constructed using parsimony and Bayesian analyses Binary feeding behavior characters were coded for all taxa in the morphological matrix and mapped onto the resulting topology using parsimony optimizations. Results from the analysis based on morphological data suggest Calpini is monophyletic and is supported by five synapomorphies. Three of these are unreversed, shared characters of the proboscis. Tearing hooks are restricted to the Calpini and little additional variation with in the tribe exists suggesting proboscis morphology may not be 13

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strongly correlated with feedi ng behavior. The results from this study support the hypothesis that hematophagy in the genus Calyptra evolved from the fruit-piercing habit as opposed to tear feeding. A preliminary checklist of Calpini is also provided, incorporating corrections and changes to publication dates and nomenclatu re as presented in recent checklists. A microbial survey of Calyptra thalictri Borkhausen using polymerase chain reaction [PCR] primers for 16S rRNA sequences for Eubact eria. High-fidelity PCR and subsequent DNA analyses indicated that at least five microorganisms belonging to the and Proteobacteria were present. Two eubacterial sequences, related to a Klebsiella sp. and a Sinorhizobium sp., were detected in the abdomens individuals sa mpled, and three additional sequences were found in some samples, suggesting all five could be a ssociated with abdominal structures. No Archaea, Fungi including yeast-like or ganisms, Microsporidia, or Wolbachia were detected. These results document the first microbial associates in a fruit-piercing and blood-feeding moth. 14

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CHAPTER 1 INTRODUCTION Literature Review This chapter reviews the basic biolog y, adult feeding behavior, and proboscis morphology of fruit-piercing and blood-feeding moths with an emphasis on moths in the tribe Calpini. The present state of the taxonomy a nd classification of Calpini is summarized and hypotheses of the evolution of feeding behaviors within the tribe are di scussed. Finally, the significance of the diversity and importance of insects and thei r microbial associations is discussed, including what is known about Lepidoptera and their microbial associates. The final portion of this chapter in troduces my research objectives, hypotheses, and how they were tested. Overview of Fruit-Piercing and Blood -Feeding Moths in Tribe Calpini The introduction of pest species to the Un ited States from 1906 to 1991 has resulted in the loss of approximately 97 b illion dollars (Pimentel et al 2005). Additional costs are associated with controlling the pests after they ha ve become established. Due to recent efforts to manage agricultural pests in a way that is environmentally re sponsible, greater emphasis has been placed on biological control and sustaina ble agricultural methods (Miller and Rossman 1995). Unfortunately some programs have been unsuccessful due to a paucity of information about the life history, taxonomy, evolutionary relationships, and basic biology of the target pests. When fundamental information about the systema tics and behavior of a group of pest organisms is lacking, biological control e fforts and agricultural system development may fail, thereby wasting time and precious resear ch dollars. Successful control programs require basic research on the pest and natural enemies so that problems with species identification, undescribed species, polymorphic species, and cryptic species can be overcome (Debach and Rosen 1991). When 15

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coupled with phylogenetic information, biosystematic studies of pest organisms can provide predictive power for identifying invasive pests and potentially enhance control efficiency. Many moth species in the insect order Lepidopter a are serious pests of crops. The largest moth family, Noctuidae, comprises 45,000 speci es (Poole 1989, Holloway et al. 2001), and includes many introduced pest species in the U.S. such as cutworms ( Agrotis Prodenia, and Euxoa ), corn earworms and budworms ( Helicoverpa ), and armyworms (Pseudaletia ). Although much attention has been paid to the taxonomy an d systematics of these moth pests and their control, negligible research has been directed at the systematics of a subgroup of these moths that are pests of agriculture, and pot entially, medical importance duri ng the adult stage: the fruit piercers and blood feeders. Within the order Lepidoptera, the ability to pierce mammalian skin and take a blood meal is restricted to the moth genus Calyptra Ochsenheimer (Lepidoptera: Noctuidae: Calpinae) (Bnziger 1968-2007, Zaspel et al. 2007, Zaspel 2008). Blood-feeding moths are subcutaneous pool-feeders, severing the capillaries below the surface of the skin in order to form a pool of blood from which they can feed (Bnziger 1971, 1980) The moths insert the tip of the proboscis into the host, cutting the tissues by moving the two straw-like tubes, or galae, of their proboscis back and forth in opposing directions; this moveme nt has been termed antiparallel motion of the proboscis (Bnziger 1971, 1980). This saw-like m ovement is often performed at short intervals followed by intermittent uptake of the blood meal. Blood-feeding moths can feed on all parts of the animals for ten minutes or longer (Zaspel et al. 2007). Penetration of the host skin by the moths barbed proboscis can be quite painful, an d the resulting wound(s) are large in diameter when compared to the wound of a mosquito bite or bee sting (Zaspel, Chapter 5). The first species recorded piercing the skin of a mammal and feeding on its blood was Calyptra eustrigata 16

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Hampson; it was discovered feeding on water buffa lo, deer, tapir, and antelope in Malaysia by Bnziger (1968). Since the descri ption of the first blood-feeding Calyptra species, males of ten additional Calyptra species have been observed piercing mammalian skin and feeding on blood under both laboratory and natural conditions (Bn ziger 1989, Bnziger 2007, Zaspel et al. 2007). While fruit feeding is obligatory in both males and females of Calyptra (Bnziger 2007), blood feeding is restricted to males a nd is facultative; they are able to survive without ingestion of a blood meal (Bnziger 2007). Males of Calyptra have been recorded feeding on a broad range of ungulate hosts and occasionally elephants and huma ns are attacked (Bnziger 2007, Zaspel et al. 2007). It has been hypothesized that the ability to pierce mammalian skin and suck blood in Calyptra spp. evolved from the fruit-piercing habit, a likely scenario given fruit-piercing and blood-feeding moths share the probos cis and behavioral modificati ons observed in both feeding types (Bnziger 2007). It is possible that th e moths may seek out mammalian hosts to obtain additional nutrients such as amino acids or s ugars, thereby increasing fitness (Bnziger 2007). Blood-feeding Calyptra males have not tested positive for proteases; however, male moths do appear to be in search of salts (Bnziger 2007). It is possible that males are sequestering salts and transferring them to the females during mating for egg production (Smedley and Eisner 1995) or to replenish salt supplies depleted during oviposition (Adler and Pearson 1982). Alternative hypotheses regarding the evolution of feeding in Calyptra have been proposed (Downes 1973, Hilgartner et al. 2007) and suggest the skin-piercing and blood-feeding behavior is derived from other animal-associated feeding beha viors such as dung, urine, or tear feeding. Although one Calyptra species, C. minuticornis (Guene), has been recorded feeding on tears (Bnziger 2007), this evolutionary trajectory is an unlikely one given the proboscis 17

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structures of fruit piercers and blood feeders are not homologous with those of tear-feeding moths, and such lachrophagous moths do not pierce fruit (Bnziger 2007). This hypothesis is also problematic given the shared behavioral modifications found in both fruit-piercing and blood-feeding moths. The tearing st ructures involved in the pierci ng of fruits or mammalian skin is restricted to a small group of taxonomically as sociated noctuid genera (Zaspel, Chapter 3), while animal-associated feeding behaviors, in cluding the imbibing of blood droplets found on the bodies of mammals, are widespread with in Lepidoptera (Bnziger 1982, Scoble 1992). These hypotheses have never been tested within an empirical phylogenetic framework, and a hypothesized directional progressi on of feeding types cannot be tested formally until the relationships of Calyptra and related genera are known. Calyptra consists of 17 described sp ecies and two subspecies (Bnziger 1983). These are medium-sized moths, with wingspans ranging fro m 35-72 mm in size. These moths typically fly during the rainy season in the tropics and in the midsummer months in temperate regions. Eggs are typically laid on the undersid e of the leaves of host plants in the Menispermaceae and the Ranunculaceae (Bnziger 1982, Fay 2005, Zaspel pers. observation). All Calyptra species have modified proboscides equipped with strongly sclerotized tearing hooks used for piercing the skin of hard fruits such as peaches and citrus, and of mammals, the number of apical tearing hooks of the proboscis varies between species and among specimens (Zaspel, Chapter 2). This variation does not appear to be in any way associated with one piercing behavior or the other (e.g., sucking fruit juices versus sucking blood) (Zaspel, Chapter 2). Systematics of Calpini The extent to which invasive species impact the environment is difficult to assess when knowledge regarding their biodiversi ty in the U.S. is lacking (P imentel et al. 2005). A major aim of systematics is to describe and organize this diversity into a classification scheme that is 18

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hierarchical, providing stability of species names that reflect history, and provi ding predictive power of relevance to biological control and pest management programs. The most recent classifications place Calyptra and other fruit-piercing genera in the subfamily Calpinae (Kitching and Rawlins 1998, Fibiger and Lafontaine 2005, Lafontaine and Fibiger 2006, Mitchell et al. 2006). Presently, Calpinae consis ts of four tribes: Anomini Grote 1882, Calpini Boisduval 1840, Phyllodini Hampson 1913, and Scol iopterygini Herrich-Schffer [ 1852] (Fibiger and Lafontaine 2005, Lafontaine and Fibiger 2006, Holloway 2005). The tribe was catalogued by Fibiger and Lafontaine (2005) wherein they a ssigned genera to the tribe, s uggesting Calpini is comprised of eleven genera. Fibiger and Lafontaine (2005) fo cused on the Palearctic region and therefore was not inclusive of all genera comprising the tribe. In addition, the fruit-piercing genus Oraesia which has proboscis armature identical to that of Calyptra and Gonodonta (Zaspel, Chapter 2), was excluded while seven genera lacking the di agnostic characteristics of the proboscis were included. All genera in Calpini contain frui t-piercing species (Fibig er and Lafontaine 2005, Holloway 2005), and a high concentration of economically important fruit-piercing species is found within the tribe. The tribe Calpini is cosmopolitan in its di stribution; however, many calpine genera have geographic distributions that ar e more restricted. The genus Calyptra is considered to be Old World in its distribution with a high concentration of diversity in South and Southeast Asia, yet one species, C. canadensis, occurs in the northeastern Unit ed States and Canada (Poole 1989, Bnziger 1989a, Fibiger and Lafontaine 2005) In the Old World tropics, species of Eudocima are common pests of hard-skinned fruits (e.g., longan), with the widespread E. fullonia (Clerck) the target of a biological c ontrol project in the region (Fay 2002, Sands and Liebregts 2005). Gonodonta species can be found in subt ropical and tropical regions, with seven species occurring 19

PAGE 20

in Florida, Texas, and Arizona; Gonodonta species pierce citrus fr uits, including tangerines, grapefruits and oranges, at times causing extens ive losses in subtropical and tropical regions (Todd 1959). Serious outbreaks of Gonodonta species occurred in Mexico and Cuba in the 1940s and 1950s, respectively, and one report from the late 1950s stated that 20 percent of the fruit in two orange groves in St. Lucie County, Florida was lost due to outbreaks of G. nutrix (Todd 1959). Oraesia and Plusiodonta species are common in the Old and New World tropics (Poole 1989, Holloway et al. 2001). Plusiodonta species have been observed feeding on fruits such as plum in South and Southeast Asia (Zaspel pers. obs. 2005). Adult Oraesia species have reportedly damaged thick-skinned fruit in Korea (Yoon and Lee 1974). Calpine larvae primarily feed on plants in the family Menispermacae. A lthough calpine larvae are not agricultural pests, all adult moths are piercers of fruits (Bnziger 1982, Holloway et al. 2001 ); punctures made in fruits by the moths cause agricu ltural loss through fermentation and rotting of the fruit or secondary invasions by microorganisms that re sult in early fruit fall (Todd 1959, Sands 1993). Proboscis morphlogy in piercing moths The structure and function of the lepidopteran proboscis has been examined for a broad range of taxa across multiple families (B nziger 1971, 1973, Bttiker et al. 1996, Krenn 1990, Krenn 1997, Speidel et al. 1996). Previous work u tilized light microscopy to evaluate proboscis structures (Hattori 1969, Bnziger 1970, 1973, 1980) followed by scanning electron microscopy (SEM) (Cochereau 1977, Bttiker et al. 1996, Spei del et al. 1996). Taxon sampling for these studies ranged from higher-level exemplars from distantly related lepidopteran families (e.g., Noctuidae, Geometridae, and Pyralidae) base d on adult feeding be havior (Bnziger 1973, Bttiker et al. 1996) to more focused proboscis studies on related speci es (Bnziger 1970, 1986), subfamilies (Speidel et al. 1996), and familie s (Krenn 1998, Krenn and Kristensen 2000, Krenn and Mhlberger 2002, Krenn and Penz 1998, Krenn et al. 2001). From this work, several 20

PAGE 21

notable differences in probosci s morphology were observed, including a wide variety of sensilla types and other specialized feeding structures (Bnziger 1970, 1973, Bttike r et al. 1996, Speidel et al. 1996). Uniquely specia lized proboscis structures (cu tting ridges, erectile barbs and eversible tearing hooks moved by blood-pressure) and concomitant stylet dynamics (anti-parallel movements, oscillatory torsion, spindle movements) occur in the piercing moths in the family Noctuidae (Knckel 1875, Breitenbach 1877, Bnziger 1970, 1973, 1980). Within the Calpinae, the tearing hooks are unique to a tribe of apparently closel y related fruit-piercing moths consisting of at least nine genera: Africalpe Krger, Calyptra Ochsenheimer, Eudocima Billberg, Ferenta Walker, Gonodonta Hbner, Graphigona Walker, Oraesia Guene, Plusiodonta Guene, and Tetrisia Walker (Zaspel, Chapter 3). The t earing structures observed in these genera arise from a socket and thus are not homol ogous with other forms of modified sensilla of the proboscis found among fruit-sucking and tear-d rinking moths within the Lepidoptera (Zaspel, Chapter 2). Blood feeding and endosymbionts in Calpini Hematophagy is believed to have arisen independently in arthr opods during the Jurassic and Cretaceous periods at leas t six, and potentially as many as 21 times (Balashov 1984, Ribeiro 1995). Adams (1999) estimates that 14,000 insect species from five orders (Pthiraptera, Diptera, Hemiptera, Lepidoptera, and Si phonaptera,) are hematophagous. All blood-feeding arthropods are capable of transmitting pathogens (Durde n and Mullen 2002). Arthropod-transmitted diseases are of great medical and veterinary significance worl dwide, and new pathogens are rapidly emerging (Gratz 1999). At least 27 major human and animal diseases are vectored by insects (Faust et al. 1962, Gratz 1999). It is known that many fruit-piercing Calyptra species and species in related genera are of great economic importance in ma ny countries, but their potential as vectors of human or animal disease rema ins unknown. Many associations between insects 21

PAGE 22

and microorganisms that are considered signifi cant are often involved in animal and plant disease transmission (Daly et al. 1998). Symbio tic relationships are defined as an organism living on or in another organism, from whic h nutrients, protection, and assistance in the metabolism of various biologica l compounds are either obtained or exchanged (Bourtzis and Miller 2006). Mutualistic symbiotic relationships involve the dependence of each organism in the system on one another for survival. In some cases, this mutualism will result in the production of unique structures a nd novel metabolisms in an arthropod host. In many insect species, endosymbiotic bacteria and even some eukaryotic microorganisms (e.g., fungi, microsporidia, yeasts) occur in the gut, sali vary glands, or reproductive tract. Although relatively little is known about the specific biological role of many insect endosymbionts, evidence suggests they are invo lved in reproduction, digestio n, nutrition, pheromone production, and protection of the insect from pathogens (Broderick et al. 2004, Dillon and Dillon 2004). Endosymbionts are found intern ally associated with th e gut, reproductive tract, mouthparts, or salivary glands of insects. Insect endosymbionts can be classified as obligatory or facultative; infection status with facultative e ndosymbionts can vary between specimens of a single species depending on the host diet or the environment in whic h the host lives (Broderick et al. 2004). Facultative relationships between th e endosymbiont and its insect host are often referred to as microbial associ ations or secondary endosymbiosis because the biological role and dependence of the host on the endosymbiont is unclear or unknown. Some microorganisms are transient inhabitants of the surface of the gut of insects and are present only sporadically or temporarily, while the host depends on obligatory or primary endosymbionts for its survival. In the aphid -Buchnera system, for example, the aphid host has specialized internal structures called mycetocytes that contain the bacteria, and the ap hid host is provided with essential amino acids 22

PAGE 23

(Moran et al. 1993). Some insects and their primary endosymbionts ar e phylogenetically linked and, in some cases, have been associated for over 200 million years (Bauman et al. 2005, Downie and Gullan 2005, Gruwell et al. 2007, Takiya et al. 2006). Relatively little is known about th e microbial associates of lepi dopteran insects. The leek moth, Acrolepiopsis assectella Zeller, harbors endosymbiotic bacteria that produce attractants in the frass that attract specific parasitoids (Thibout et al. 1995 ). Broderick et al. (2006) demonstrated the biological insecticide Bacillus thuringiensis (Bt) is ineffective against gypsy moth larvae, Lymantria dispar (L.), in the absence of their normal midgut microbiota, and the insecticidal activity of the Bt was restored when the naturally occurring bacteria were reintroduced to aposymbiotic hosts. Other stud ies with Lepidoptera have primarily focused on the occurrence of Wolbachia and the role it plays in reproduct ion and distortion of sex ratios. Within butterflies and moths, Wolbachia has been correlated with reproductive anomalies such as male-killing, cytoplasmic incompatibility, and se x-role reversal in ma les (Jiggins et al. 1998, 2000, 2001). Wolbachia is prevalent in both butterfly and moth populations in some regions; Tagami and Miura (2004) surveyed 49 species (500 individuals) in nine lepidopteran families in Japan for Wolbachia, and found seven of nine families (78%) and 22 of 49 (44.9%) species were positive. Few studies have focused on the complete mi crobial community of lepidopteran hosts. Broderick et al. (2004) surveyed the midgut of third-in star gypsy moth larvae from laboratory and field populations in order to better understa nd the relationship between a lepidopteran host and its midgut associates. The effects of diet on the bacteria in the midgut were examined using culture and culture-independent methods using larv ae reared on four differ ent tree hosts, and on an artificial diet. A total of 23 eubacterial strains, or phylotypes were detected among the 23

PAGE 24

larvae sampled, and it was determined that the eubacterial diversity present in individual larvae was dependent on the diet. These bacteria coul d be derived from epiphytic bacteria found on foliage or from other environmental sources and may be transient inhabi tants of the gut. Enterobacter sp. and E. faecalis, which are typically found in inse ct guts, were present in all larvae, suggesting an important role in gypsy moth biology (Brode rick et al. 2004). A complete understanding of the biology and feeding behaviors of fruit-piercing and blood-feeding moths requires basic knowledge of both their internal and external environments. This includes, but is not limited to detection and identification of the microbes associated with these moths. Recent findings suggest that natu rally occurring gut microbes play an important role in the ability of insect vectors to transm it disease to their host (S t. Andr et al. 2002). Studies have shown insect vector capabilities to be highly infl uenced by the composition of the microorganisms found naturally in the midguts of some vectors (Dillon and Dillon 2004). While it is unclear whether these moths ingest microbial parasites during the blood meal, the survival of disease organisms in Calyptra species could depend on the pres ence of other microorganisms in the midgut. Research Objectives Chapter 2. The primary goal of this chapter was to survey the structures associated with the proboscis in fruit-piercing, blood-feeding, and tear-feeding moths of the subfamily Calpinae using both light microscopy and scanning electr on micrograph (SEM) methods. I hypothesized that specific proboscis structures in piercing moths could be a ssociated with specific adult feeding behaviors. The secondary goal of this study was to homologize the structures of the proboscis across the subfamily using exemplar ta xa selected from available checklists and faunistic guides. The resulting products of this study will include complete descriptions of structures for all included calpine taxa and glossary of terms for proboscis morphologies; 24

PAGE 25

presently, no such glossaries are av ailable. This study will also be the first to compare structures visable using light microscope imaging and SEM imaging. Finally, this study will be used to determine whether proboscis morphology can be us ed for reconstructing a natural classification and predicting differences in adult feeding be havior in these moths by directly comparing proboscis structures of taxa in each assigned feeding category. Chapter 3. Previous research on the proposed relationships between calpine genera was not based on formal analyses of empirical data and no phylogenetic hypotheses for the tribe Calpini are available. Thus, the primary objectiv e of this chapter was to reconstruct a phylogeny of Calpini using morphological and molecular data The resulting phylogeny will be used to test the hypothesis of a directional progr ession of feeding types from n ectar feeding to fruit piercing to skin piercing and blood feeding in these calpine moths. The specific questions addressed by this research were as follows: 1) What are th e evolutionary relationships among the genera in Calpini? 2) Are the genera within Calpin i monophyletic, and which mo rphological characters can be used to diagnose clades within the tribe? 3) How many origins of blood feeding occurred in Calyptra ? 4) Is there a directional pr ogression of feeding types in these moths? 5) Is feeding behavior correlated w ith mouthpart morphology? The evolutionary relationships of fruit-piercing and blood-feeding moths in the tribe Calpini will be investigated using the followi ng character systems: h ead appendages including the surface microstructure(s) of the proboscis, abdominal sternite s and tergites, and male/female genitalia. In addition, data from one mitochondrial gene (COI) and a fragment of the nuclear large subunit (28S, D2 region) rRNA will be used. This study will document the first test of the hypothesis of a directional progression of feeding t ypes in Lepidoptera. This study also formally tested whether proboscis morphology is correlated with feeding be havior in frui t-piercing and 25

PAGE 26

blood-feeding moths (e.g., presence of tearing hooks with primary fruit-piercing of hard skinned fruits, shape of sensilla styloconica with blood feeding, and position of erectile barbs with secondary piercing of hard skinned fruits). Chapter 4. The tribe Calpini was recently catalo gued by Fibiger and Lafontaine (2005) wherein they assigned additional genera to the tribe, suggesting Calpini consisted of approximately 200 species in 11 genera. This pu blication focused on the Palearctic region and was therefore not inclusive of all genera compri sing the tribe. In a ddition, the fruit-piercing genus Oraesia Guene, which has proboscis arma ture identical to that of Calyptra and Gonodonta (Zaspel, Chapter 2), was excluded while seven genera lacking these diagnostic characteristics of the proboscides were included. The primary objective of this chapter was to combine the works of Holloway (2005) and Fibi ger and Lafontaine ( 2005) into an updated checklist to complement recent taxonomic st udies (Bnziger 1983, Zilli and Hogenes 2002), a survey of calpine proboscis morphology (Zaspel et al. 2008), and phylogenetic research (Zaspel, Chapter 3). This checklist also serves to correc t minor taxonomic errors in the checklist of Poole (1989). All original descriptions for each genus species, subspecies, and their synonyms were studied. The checklist includes t ype localities if available, a complete references list, and corrections and changes to the nomenclature pres ented in the checklists of Poole (1989), Fibiger and Lafontaine (2005), and Holloway (2005). Chapter 5. Comparative studies elucidate evolu tionary trends by comparing certain characteristics, i.e. descriptions of the e nvironments inhabited by the organisms, phenotypic characters, and behaviors acro ss taxa represented in a phyloge ny (Harvey and Pagel 1991). Comparative analyses of feeding behaviors require that both the behaviors and hosts of the fruitpiercing and blood-feeding moths for each taxon are known (Harvey and Pagel 1991). 26

PAGE 27

Acquisition and characterization of feeding behavior data for the moth species under investigation was the primary goa l of this study. Of the 17 known Calyptra species (Bnziger 1983), C. eustrigata (Hampson), C. minuticornis minuticornis (Guene), C. orthograpta (Butler), C. bicolor (Moore), C. fasciata (Moore), C. ophideroides (Guene), C. parva Bnziger and C. pseudobicolor Bnziger have been reported to pierce ma mmalian skin, with the latter five also able to pierce humans, under natural conditions. Calyptra fletcheri (Berio) has pierced humans in experiments (Bnziger 1968, Bnziger 1989). These species are considered facultative or opportunistic blood-feeders primarily in subtropical areas in southern Asia and tropical Southeast Asian countries (Bnziger 1989b) At least four additiona l closely related genera ( Eudocima Gonodonta, Oraesia and Plusiodonta ) have apparently homologous proboscides modifications used for fruit-piercing, but the occurrence of blood-feeding in those species has not been observed (Bnziger 1979, Zaspel, unpublished data). The purpose of this chapter was to document the feeding behavior of two species ( C. thalictri and C. lata ) occurring in a temperate region under experimental and semi-natural cond itions to determine whether they were also facultative blood feeders or strictly fruit piercers. Chapter 6. Until recently, the moth Calyptra thalictri was considered to be an obligate fruit piercer that never took blood meals from mammals. On an expedition to the Primorye Territory of Far Eastern Russia, sp ecimens from a remote population of C. thalictri were documented piercing human skin and feeding on the blood for the first time, suggesting these moths will feed on mammalian hosts when fruits are unavailable (Zaspel et al. 2007). This chapter surveys males of C. thalictri collected in Russia in 2006 for potential microbial associates and discusses their potential biologi cal role. This moth species provides a recent example of the acquisition of hematophagic be havior within the Le pidoptera. Heads and 27

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abdomens of adult males of fruit-piercing and blood-feeding C. thalictri were used to determine whether or not microorganisms are associated with C. thalictri I hypothesized that bloodfeeding moths could be associated with microorganisms that were known animal pathogens, or could potentially play a biological role in its insect host. The re sults from this chapter document the first case of microbial associates in a fruit-piercing and bl ood-feeding moth. Chapter 7. This chapter focuses on storage methods for future amplification of endosymbiont DNA. Understanding adequate ti ssue preservation methods was important for fieldwork during my dissertation and for obtaining specimens us ed in Chapters 3 and 6. Methods proposed for the preservation of insect tissue for DNA analysis have included various concentrations of ethanol, Carnoys solution, liquid nitrogen, and acetone (Post 1993; Dessauer 1996; Fukatsu 1999; Mtambo 2006). However, little attention has been paid to appropriate storage methods for future detection of endosymbi ont DNA within an insect host (Fukatsu 1999). Some studies report successful amplification of bacterial DNA in a host af ter thousands of years (Salo et al. 1994; Fricker et al. 1997; Willers lev et al. 2004), but others have reported inconsistent amplification of bacterial DNA due to low titers of the bacteria in the host, difficulties with the DNA extraction process, PCR-i nhibiting substances present in the insect gut, or storage method (Fukatsu 1999; Barnes et al. 2000; Bextine et al. 2004 ; Hoy and Jeyaprakash, unpublished data). Fukatsu (1999) suggested acet one storage was superior to ethanol as a preservation method for both the amplifica tion of insect host DNA and the DNA of their endosymbionts. The goal of this chapter was to compare molecula r methods for the detection of Wolbachia in the Mediterranean flour moth Ephestia kuehniella (Keller) (Lepidoptera: Pyralidae), and potentially other endosymbiotic b acteria in their insect host, in preserved specimens over time. I hypothesized that the ability to amplify endosymbiont DNA in a 28

PAGE 29

lepidopteran host would decline over time depe nding on both the method of storage and method of DNA amplification. For example, after two months storage in ethanol at -80C, Wolbachia DNA could not be amplified consistently using high fidelity PCR in the honeybees Apis mellifera scutellata and A.m. capensis (Hoy and Jayaprakash, unpublished observation). Standard, high-fidelity (HF), and real-time quantitative (RTQ) PCR methods were used to detect and quantify Wolbachia DNA from E. kuehniella specimens stored under 4 treatment conditions (2 in 95% EtOH and 2 in acetone) over a 2-year storage period. Spectrophotometry readings were taken at each assay (n = 9 over a 2-year period) to ensure consistency of concentration and quality of template DNA for each treatment. Stored samples were compared to fresh specimens at the end of the experiment. Chapter 8. Finally, in this chapter, entitled Perspect ives, I evaluate my experience as a Ph.D. student in the Department of Entomology and Nematology at the University of Florida. This chapter is meant to be reflective and considers what I learned and what I might do differently in the future. 29

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Figure 1-1. Calyptra thalictri male feeding on human thumb (JMZ) in Russia 2008. 30

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CHAPTER 2 A COMPARATIVE SURVEY OF PROBOSCIS MORPHOLOGY AND ASSOCIATED STRUCTURES IN FRUIT-PIERCING AND BLOOD-FEEDING MOTHS IN THE SUBFAMILY CALPINAE (LEPIDOPTERA: NOCTUIDAE) Introduction The structure and function of the lepidopter an proboscis has been examined for broad range of taxa across multiple families (B nziger 1971, 1973, Bttiker et al. 1996, Krenn 1990, Krenn 1997, Speidel et al. 1996). Previous work u tilized light microscopy to evaluate proboscis structures (Hattori 1962, Bnziger 1970, 1973, 1980) followed by scanning electron microscopy (SEM) (Cochereau 1977, Bttiker et al. 1996, Spei del et al. 1996). Taxon sampling for these studies ranged from higher-level exemplars from distantly related lepidopteran families (e.g., Noctuidae, Geometridae, and Pyralidae) base d on adult feeding be havior (Bnziger 1973, Bttiker et al. 1996) to more focused proboscis studies on related speci es (Bnziger 1970, 1986), subfamilies (Speidel et al. 1996), and familie s (Krenn 1998, Krenn and Kristensen 2000, Krenn and Mhlberger 2002, Krenn and Penz 1998, Krenn et al. 2001). From this work, several notable differences in probosci s morphology were observed, including a wide variety of sensilla types and other specialized feeding structures (Bnziger 1970, 1973, Bttike r et al. 1996, Speidel et al. 1996). Uniquely specia lized proboscis structures (cu tting ridges, erectile barbs and eversible tearing hooks moved by blood-pressure) and concomitant stylet dynamics (anti-parallel movements, oscillatory torsion, spindle movements) occur in the piercing moths in the family Noctuidae (Knckel 1875, Breitenbach 1877, Bnzig er 1970, 1973, 1980), with a majority of the taxa currently placed in the Calpinae. The subfamily Calpinae presently consists of four tribes (Anomini, Calpini, Phyllodini, and Scoliopterygini; Fibiger and Lafontaine 2005, Holloway 2005) and is defined by the tearing structures of the proboscis (Goater et al. 2003, Fibiger and Lafontaine 2005, Holloway et al. 31

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2001, Holloway 2005, Kitching and Rawlins 1998). Calpinae is cosmopolitan in its distribution; however, many genera have geographic distributions that are somewhat restricted. Species in the subfamily exhibit a broad range of adult feeding behaviors including those that can be considered piercers of fruits or other hos ts. It has been known for some time that fruit piercers can be primary or secondary depending on whether they are able to penetr ate intact skin, or only fruit damaged previously by primary piercers or othe r animals, respectively (Jack 1922). Bnziger (1982) proposed a more precise ch aracterization of which moths can pierce what type of fruits (see below). Within the tribe Calpini, males of ten Calyptra species pierce mammalian skin and feed on blood (Bnziger 1971, 1982, 1989, Zaspel et al. 2007, Zaspel 2008). Some remaining species appear to be exclusively fruit pi ercing; however, also hematophagous Calyptra are obligatory fruit piercers in South and Southeas t Asia (Bnziger 2007). Species of Eudocima are common pests of a wide variety of fruits ranging from hardskinned longa n, to thick-skinned oranges, to soft or ripening fruits (e.g., peaches, plums, apples; (Fig. 2-1D), with the widespread Eudocima fullonia (Clerck) being the ta rget of a biological control proj ect in the region (Fay 2002, Sands and Liebregts 1993). Gonodonta species can be found in subtropical and tropical regions, with seven species occurring in Florida, Texas, a nd Arizona; these species pierce citrus fruits, including tangerines, grapefruits and oranges, at times causing extensive losses in subtropical and tropical regions (Todd 1959). Other species, such as Calyptra thalictri (Borkhausen) (Fig. 2-1F) and non-calpini Scoliopteryx libatrix L. (Fig. 2-1C), Ophiusa tirhaca (Cramer), and Dysgonia algira (L.), pierce fruit even in temp erate Europe (Bnziger 1969, 2007). Plusiodonta and Oraesia species are common in both the Old a nd New World tropics (Poole 1989, Holloway et al. 2001). Plusiodonta species have been observed feeding on soft-skinned fruits (peaches and 32

PAGE 33

plums) in South Asia (Nepal: Ka thmandu valley, pers. observation 2005). Oraesia species cause damage to thickand soft-skinned fruit in Indi a, Nepal (Fig. 2-1A), Thailand, Korea and Japan (Ramakrishna Ayyar 1944, Hattori 1962, Yoon and Lee 1974, Bnziger 1982, 1987). Punctures made in fruits by the moths cause agricultural lo sses through fermentation and rotting of the fruit or secondary invasions by vari ous microorganisms that result in early fruit fall (Todd 1959, Sands and Leibregts 1993). Phylogenetic analyses indicate that Calp ini is monophyletic and is supported by the presence of tearing hooks of the proboscis (Zaspel, Chapter 3). At least three genera currently placed in the Calpini and others in related tribes lack tearing hooks, confusing the classification of the subfamily and the tribes th erein. The taxonomy of Calpini a nd associated genera is further complicated by the occasional inclusion of apparently tear-feeding taxa (e.g., Hemiceratoides ; Fig. 2-1E) and the assertion that the tear-feeding hab it evolved from the fruit feeding habit (Hilgartner et al. 2007). Th ere is no evidence yet that Hemiceratoides is fruit piercing, nor that tear drinking is its normal feeding habit; so fa r, there are more compelling arguments that skinpiercing blood-sucking in mammal s evolved from fruitpiercing while tear dr inking developed in separate lineages (Bnziger 1980, 2007). Bnziger (2007) pointed out discrepancies in Bttiker et al. (1996) and Hilgartner et al. (2007). Previous worker s describing the proboscis morphology of H. hieroglyphica and associated structures of have applied terms incorrectly (Hilgartner et al. 2007); further, no specific criteria fo r the application of the terms or glossary that can be used in describing proboscis morphology exists. The primar y focus of this study was to compare and describe the diversity of probosci s structures across the genera cu rrently placed in the subfamily Calpinae using both SEM and light microscopy, a nd to accurately homologize these structures within the subfamily. 33

PAGE 34

Materials and Methods Terminology The surface microstructure and visible macro-st ructures of the proboscis in calpine moths are described using light micros copy and SEM imaging; comparisons are made between the two imaging methods for viewing probos cis structures within the Lepidoptera. The terminology used herein for proboscis morphology follow the work of Bnziger (1970, 1980), Bttiker et al. (1996) and Speidel et al. (1996). Sensilla te rminology follows Altner and Altner (1986), Faucheux (1985, 1991, 1995), Hallberg (1981), and Hallber g et al. (1994). A list of definitions and criteria for the various sensi lla and other proboscis structures examined is provided in Table 2-1. A proboscis diagram illustrating proximal ve rsus apical regions for calpine moths is provided ( Oraesia rectistria Fig. 2-2). Because there was little structural variation within a genus, exemplars of the genera surv eyed are figured and discussed. Proboscis morphologies for repr esentative calpine genera included in the survey are discussed according to functiona l feeding group. Func tional feeding groups were characterized based on Bnziger (1982) and Norris (1935). Fo r the sake of conveni ence, Bnziger (1982) chose four categories of fruits based on their increasing difficulty to be pierced by moths: very soft-skinned fruit (e.g. raspberry), soft-skinned fruit (e.g. peach, grape), thick-skinned fruit (citrus), and hard-skinned fruit (longan, lichi). Moths were grouped according to their ability to pierce the four categories as primary piercers. For example, a moth like Calyptra minuticornis is a primary piercer of thick-skinne d fruits (oranges) and all softer-s kinned fruit, but a secondary piercer of hard-skinned fruit (longan). The fi rst two categories are omitted here because the genera analyzed belong only to the latter two, unlike those st udied by Bnziger (1982) that included the catocalines that mostly have far weaker and less armored proboscides. The other feeding types, i.e. non-piercing fruit sucking, nectar sucking, non-piercing blood sucking, skin 34

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piercing blood sucking, and the va rious degrees of lachryphagy, were characterized in Bnziger (1973, 2007). Some feeding groups include taxa that exhibit polyt ypic feeding behaviors; thus, observed continuity or overlap between feeding types will be discussed for those taxa. The following is a list of the institutional and privat e collections consulted during this study. The acronym of the institution or name of private collection is followed by the name of the individual that prepared the loan. Acronyms follow Heppner and Lamas (1982): AMNH American Museum of Natural History, New York (T. Schu h); HB Personal collection of H. Bnziger, Thailand (Hans Bnziger); MF Personal collection of M. Fibi ger, Denmark (Michael Fibiger); FLMNH Florida Museum of Natural Histor y, Florida (G. Austin, M. Thomas); NMNH National Museum of Natural Histor y, Washington D.C. (M. Pogue). Characterization of Functional Feeding Catergories Non piercing, fruit sucking. The moths in this feeding gr oup obtain nutr ients through various fruit juice sources. The moths in this category take up fru it juice from cracked or damaged fruits; no piercing is involved. While fruit sucking is a common phenomenon within the Lepidoptera, the taxa sampled in this study were limited to moths in the following calpine genera (sensu Fibige r and Lafontaine 2005): Goniapteryx servia (Stoll), Hypsoropha hormos Hbner, Phyprosopus callitrichoides Grote. Primary piercers of thick-skinned fruit but secondary piercers of hard-skinned fruit. Moths in this category can pier ce the intact rind of thick-skinned fruit such as mandarin, as well as all softer-skinned fruit, but not hard-s kinned longan and, by extension the related lichi (not studied by us). However, softer varietie s of longan and lichi possi bly may occasionally be pierced by some of the larger moths that have stronger proboscides (e.g. Calyptra orthograpta, Oraesia emarginata). Conversely, some genera, such as Plusiodonta and Anomis (Fig. 2-1B) (the proboscis of which lacks the tearing hooks), have smaller proboscides and may not always 35

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be able to penetrate mandarin. Many noctuid lineages have been described as fruit-piercers (Bnziger 1982; Yoon and Lee 1974). The following taxa were examined representing the tribe Calpini (sensu Fibiger and La fontaine 2005; Holloway 2005): Calyptra albivirgata (Hampson), C. bicolor (Moore), C. canadensis (Bethune), C. eustrigata (Hampson), C. fasciata (Moore), C. fletcheri (Berio), C. gruesa (Draudt), C. lata (Butler), C. minuticornis (Guene), C. ophideroides (Guene), C. orthograpta (Butler), C. parva Bnziger, C. pseudobicolor Bnziger, C. subnubila (Prout), C. thalictri (Borkhausen), Gonodonta nutrix (Cramer), Plusiodonta coelonota (Kollar), P. compressipalpus Guene, P. incitans (Walker), Oraesia argyrosigna Moore, O. emarginata (Fabricius) O. excavata (Butler), O. excitans Walker, O. glaucochelia (Hampson), O. honesta Walker, O. nobilis Felder and Rogenhofer, O. provocans Walker, O. rectistria Guene, O. serpens Schaus, O. striolata Schaus, O. triobliqua (Saalmller), as well as two genera representing the calpine tribes Anomini and Scoliopterygini: Anomis mesogona (Walker), A. privata (Walker), and Scoliopteryx libatrix (L.) (sensu Fibiger and Lafontaine 2005; Holloway 2005). It shoul d be noted that while Scoliopteryx libatrix is likely to be able to pierce the sound rind of mandarin, so far this has not yet been studied. It is a confirmed primary piercer of soft-skinned fruit (Bnziger, 1969, and unpubl .). It is uncertain at this time whether these genera are taxonomically misplaced or if the feeding behavior is the result of ecological convergence. Presently, the piercing genera placed in the Calpini form a monophyletic assemblage (Zaspel, Chapter 3), and their associat ed proboscis modifications are restricted to the taxa therein (e.g., tearing hooks a nd sensilla styloconica dorsoventr ally flattened into erectile barbs, both of which are movable by blood pressure; Bnziger 1980). Primary piercers of hard-skinned fruit. This feeding group pierces intact skin of longan and lichi, as well as all softer-skinne d fruit. The following taxa were examined 36

PAGE 37

representing the tribe Calp ini (sensu Fibiger and Laf ontaine 2005; Holloway 2005): Eudocima homaena (Hbner) and E. salaminia (Cramer). Mammalian skin piercers and blood sucking. Of all butterflies and moths, only ten species from one genus, Calyptra have been observed using their proboscis to pierce mammalian skin and imbibe blood. Except for some Calyptra species that are fruit pests in orchards, the hematophagous species are uncommon to rare in nature and thus blood-feeding occurrences are also rarely seen (Bnziger 2007). It should be noted that the females are not hematophagous, that the males are facultatively he matophagous, that they ca nnot digest proteins but sequester NaCl (though some unknown component may also be utilized) (Bnziger 2007). Bnziger (1986, p 122-123 and Table 6) gave de tailed data on the proboscis length, width, number and length of both teari ng hooks and erectile barbs of C. eustrigata, C. minuticornis, C. orthograpta and C. fasciata The differences are clear enough to allow a rough identification of the four species based solely on the armature. Nevertheless, he noted that the armature is essentially the same and all can pierce mammalian skin. Two of the species, C. thalictri and C. fasciata exhibit differential feeding behaviors de pending on their geographic region (Bnziger 1989; Zaspel et al. 2007); also, C. fletcheri and C. thalictri, so far, have only been hematophagous under experimental or semi experimental conditions (Bnziger 1989, Zaspel et al. 2007, Zaspel 2008a), but for convenience are pl aced in this category. The remaining moth species (Calyptra bicolor, C. eustrigata, C. fascia ta, C. minuticornis, C. ophideroides, C. orthograpta, C. parva, and C. pseudobicolor ) have been recorded feeding on blood both in either natural and or laboratory conditions. The probosci des of these species were examined in this study. 37

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Tear drinking. Lachrymal fluid feeding occurs in at least six lepidopteran families: Geometridae, Notodontidae, Noctuidae, Pyrali dae, Sphingidae and Thyatiridae (Fig. 11; Lobacraspis griseifusa at batend eye; Bnziger, 1973, 1992; Bttiker 1973; Bttiker et al. 1996; Norris 1935). Most moths in this feeding group place the distal part of the proboscis onto the eyelid where it joins the eye and imbibe tears from the host. The Noctuidae have the most advanced lachryphagous species, e.g., Arcyophora spp. and Lobocraspis griseifusa Hampson, but they belong to Nolinae and are, like all othe r confirmed tear drinkers neither fruit piercing nor skin piercing blood sucking, and do not have piercing armature (Bnziger 1973). However, a recent study (Hilgartner et al. 2007) reported a moth species from a genus previously associated with the genus Calyptra by Karsch (1896) apparently drinking fluid from the eyes of a sleeping bird in Madagascar. This species, Hemiceratoides hieroglyphica (Saalmller), has some proboscis structures that are superf icially similar to those of the piercing species in the Calpini; however, it did not place within the Calpini in a preliminary phyl ogenetic analysis of the tribe based on DNA and morphological characters (Zas pel, Chapter 3). Despite its uncertain phylogenetic placement at this time, it is possible th at this species is a member of the Calpinae and might represent an independent tear-f eeding origin for the subfamily. Given Hemiceratoides previous association with other calpin e taxa and apparently unique proboscis modifications, we have included this species and a non-calpine clas sical tear drinker, L. griseifusa for comparison in the tear-drinking category in our treatment of the proboscis morphologies. Uncertain Taxa. Ferenta spp., Graphigona regina (Guene) and Tetrisia florigera Walker are placed in this category because their piercing capabilities have not yet been reported or tested. The proboscides of species in these th ree genera are virtually identical to that of 38

PAGE 39

species in Eudocima and so we speculate that the species are piercers of hard -skinned fruits, but until this can be confirmed their feeding behaviors are considered uncertain. The fourth tribe in this su bfamily, Phyllodini (sensu Holloway 2005), is represented in this survey by a single taxon: Phyllodes consobrina Westwood. Bnziger (1982) found Phyllodes consobrina and P. eyndhovii piercing Ficus mandarin, and rambutan but it was not clear whether as primary or secondary piercers. They were conservatively assessed as primary piercers of soft-skinned fruit but it is likely that they can pi erce sound thick-skinned mandarin. Species in this tribe are rare, a nd observations of their feeding behaviors are scarce. It has been suggested that the tribe Phyllodini is synonymous with the Calpini (Fibig er pers. communication; Speidel et al. 1996b), but this has not been test ed formally through phylogenetic analysis due to the unavailability of male-femal e taxon pairs for morphological anal ysis or fresh material for molecular analysis at this time. Specimen Preparation Microptics imaging Proboscis preparations of male and female individuals of 42 species from twelve genera representing the four tr ibes currently placed in the Calpinae (Fibiger and Lafontaine 2005, Holloway 2005) were includ ed in this study (Table 2-2). Dried proboscides were removed at the base of the head from pinned specimens using a fine-tip forceps and were submerged in 10% cold KOH for 18 hours, followed by short heating treatments (30 min.). Structures were cleaned in severa l rinses of approximately 70% ethanol (Winter 2000). Structures were then placed in a watch gl ass positioned with K-Y jelly, and covered with 70% ETOH for digital imaging. All digital images (96 total) were taken on a Microptics (Visionary Digital) laboratory workstation with a Nikon D1 dig ital SLR camera, using a K2 microscope lens fitted with a CF-3 objective when photographing whole structures (p roximal region proboscis shots); the K2 lens 39

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fitted with a 10X objective was used for the di stal region proboscis shots. Permanent slide mounts (Euparol [Bioquip, Garden City, CA]) were made of all probos cides. Slides were placed on slide driers for 24 48 hours and then cured for 6-months while horizontal in a slide cabinet. SEM imaging. Due to a lack of variation in pr oboscis features within each genus, proboscis preparations of both male and female specimens of 17 species representing the same 12 genera were selected as exemplars from the li ght microscope samples and were used to take scanning electron micrographs (Table 2-2). Probos cides were removed at the base of the head from pinned specimens using a fine-tip forcep s and were submerged in 10% cold KOH for 18 24 hours, followed by heating treatments (30 min.). Structures were cleaned in several rinses of approximately 70% EtOH (Winter 2000). Structures were dehydrated ov ernight in 95% EtOH, then further dehydrated using a critical point dryer and positi oned on the SEM viewing stubs. Once mounted, the samples were sputter coated using a Denton Vacuum Desk III LLC sputter coater (Moorestown, NJ) for 2-4 minutes. A to tal of 92 images were taken using a JSM-5510LV scanning electron microscope (JOEL, USA). As pointed out in Bnziger (1971, 2007), drying tends to generate unequal shrinking of the probos cis. This is slight in sclerotized piercing proboscis but can be so strong in soft, non-pierci ng proboscis to become deformed (cf. Figs. 24). While this seriously impairs a correct und erstanding of how a proboscis and its armature work in a live insect, here we essentially study only the shape (not position) of the sensilla, barbs, hooks, etc. which are not deformed by drying. 40

PAGE 41

Results Description of the piercing structures of the proboscis visible by light microscopy The basic characteristics of the surface microstructure and most other structures of the proboscis in calpine moths can be visualized using a light microscope. Six proboscis images taken using the Microptics system were selected from the 96 total images and used to describe associated structures (Figs. 2-3A-3F). The proboscis mor phology is described for a group of selected taxa examined in the Primary piercers of thick-skinned fr uit but secondary piercers of hardskinned fruit: O. serpans. The surface of the proximal region of the proboscis is simple and smooth with circular or semi-circular ribs. Chaetiform sensilla and other cuticular processes are not visible or are absent. Sens illa styloconica are modified into dorsoventrally flattened, ovate erectile barbs (eb) (Fi g. 2-3A). The erectile barbs are abundant in the apical region of the proboscis, along with distinct rasping spine-like structures. The rasping spines are triangular and without a visible distal connus. The ventral surface of the apical region is smooth and the tip bears tear-shaped, socketed tearing hooks (th) (Fig. 2-3B). Furcate sensil la are present along the lateral margin of the dorsal galeal crosslinkage (Fig. 2-3B). Proximal and apical regions are without visible sensilla basico nica or sensilla trichoidea. The ligulae of the dorsal galeal crosslinkage are triangular (Fig. 2-3B). G. indentata. The surface of the proximal region of the proboscis is simple and smooth w ith circular or semi-circular ribs (Fig. 2-3C). Chaetiform sensilla and other cuticular proc esses are not visible or are absent. Sensilla styloconica are modified into dorsoventrally flattened, ovate erectil e barbs (eb) (Fig. 2-3C). The erectile barbs are abundant in the apical region of the probos cis, along with distin ct rasping spine-like structures. The ventral surface of the apical region is smooth and the tip is with cone-shaped, socketed tearing hooks (th) (Fig. 23C). Furcate sensilla are pres ent along the late ral margin of the dorsal galeal cros slinkage. Sensilla basiconica or sensil la trichoidea are not visible in either 41

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proximal or apical regions. The ligulae of the dorsal galeal crosslinkage (dgl) are triangular (Fig. 2-3C). Primary piercers of hard-skinned fruit: E. homaena. The surface of the proximal region of the proboscis is simple and smooth with di agonal circular or semi-c ircular ribs. Two or three dorsoventrally flattened, tria ngular rasping spines (rs) occu r just below the junction of the ribbed and smooth area of the apical region (Fig. 2-3D). Erectile barbs in depressions linked by endocuticula occur just below the junction of the ribbed and smooth areas of the apical region. The surface of the apical region is smooth with serrated ridges (sr) The ventral surface of the apical region is smooth and the tip is with so cketted tearing hooks (th) linked by pale, elastic endocuticula (2-3D). The ligulae (l) of the dorsa l galeal crosslinkage (dgl) are long and spikelike (Fig. 2-3D). Mammalian skin-piercers and blood sucking: C. fasciata The surface of the proximal region of the proboscis is simple and smoot h with circular or semi-circular ribs (Fig. 23E). Chaetiform sensilla and other cuticular pro cesses are not visible or are absent. Sensilla styloconica are modified into dorsoventrally flattened, ovate erec tile barbs (eb) (Fig. 2-3E). Individual erectile barbs are positioned in a singl e row along the lateral side of the proboscis but in the apical region they are present on all sides, lacking only in the tip re gion (Fig. 2-3E). They are distally inclined and set in endocuticular depressions when at rest but, for piercing, they bulge out by blood pressure, turning the barbs into a proximally inclined position. Furcate sensilla are present along the lateral margin of th e dorsal galeal crosslinka ge (dgl) (Fig. 2-3E). The apical section is lance-like, fully scleroti zed and hence stiff. Th e ventral surface of the apical region is smooth and dorso -laterally the tip bears curved tear-shaped, tearing hooks (th) (Fig. 2-3F). They are surrounded by (pale) elastic endocuticula and set in a circular sclerotized socket. This is more protruded distally to form a collar that prevents overturning of the tearing hooks. Sensilla basiconica or sens illa trichoidea are not visible in either proximal or apical 42

PAGE 43

regions. The ligulae of the dorsal galeal crosslin kage are curved and triangular (Fig. 2-3F). Tear drinking: L. griseifusa The surface of the proximal region of the proboscis is simple and smooth (Fig. 2-4A) with minute tria ngular spines present (mts). Sensilla styloconica (ss) are present and with a distal connus (F igs. 2-4A and 2-4B). Sensilla basiconica or sensilla trichoidea are not visible in either proximal or apical regions. The ligulae of the dorsal galeal crosslinkage (dgl) are triangular and are incu rved (Fig. 2-4A). The apex of the proboscis is pale in appearance, membraneous and blunt (Fig. 2-4B). Description of the structures visible by scanning electron microscopy The proboscis morphology is described in detail for a group of selected taxa examined in each of the five functional feeding groups by scanning electron microscopy. As pointed out in Bnziger (1971, 2007), drying tends to generate unequal shrinking of the proboscis. This is slight in a sclerotized pierci ng proboscis but can be strong in a so ft, non-piercing pr oboscis to as to become deformed (cf. Figs. 2-4). While this se riously impairs a correct understanding of how a proboscis and its armature work in a live insect, here we essentially study only the shape (not position) of the sensilla, barbs, hooks, etc., which are not deformed by drying. The proboscides of selected spec ies from genera placed in the Non-piercers fruitsucking: G. servia The surface of the proximal region of th e proboscis is fluted with circular ribs and distinct longitudinal de pressions throughout (Fig. 2-5A). Cuticular processes are absent from the ribs. Sensilla styloconica (ss) are ab sent from the proximal region (Fig. 2-5A). The apical region is densely nodulose with asym metrical nodules throughout (Fig. 2-5B). The nodules near the ligulae of the dorsa l galeal cross linkage are separated by se pta (Fig. 2-5B). Sensilla styloconica (ss) are present in the api cal portion of the probosci s, with each sensillum consisting of a stylus with longitudinal ridges and an apical sensory cone (sc); proximal and apical regions are without visible sensilla basiconi ca, sensilla trichodea, or cuticular processes. 43

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The ligulae (l) of the dorsal galeal crosslinkage (dgl) are primarily triangluar, with most terminal ligula slightly twisted, with two lateral prominences (Figs. 2-5A and 2-5B). H. hormos The surface of the proximal region of the proboscis is fluted and nodulose with longitudinal depressions faintly present in most proximal circular ribs (1/4 entire leng th of proboscis) (Fig. 25C). Sensilla trichoidea or othe r cuticular processes are absent from the ribs. The apical region is densely nodulose with the nodules of the apic al region asymmetrical and separated by septa toward the apex (Figs. 2-5C and 2-5D). Sensill a styloconica (ss) are di stributed throughout the proximal and apical portions of the proboscis, w ith each sensillum consisting of a stylus with longitudinal ridges and an apical sensory cone (sc); proximal and apical regions are without visible sensilla trichodea or cuti cular processes (Figs. 2-5C and 2-5D). The ligulae (l) of the dorsal galeal crosslinkage (dgl) are rectangular, slightly twis ted, terminating in two lateral prominences (Fig. 2-5C). P. callitrichoides. The surface of the proxima l region of the proboscis is fluted with circular ribs and distinct longitudinal depre ssions throughout (Fig. 2-5E). Cuticular processes are absent from the ribs. Sensilla styloconica are absent from the proximal region (Fig. 2-5E). The apical region is sparsely nodulose becoming densely nodulose towards the apex of the proboscis (Fig. 2-5F). Nodules of the apical region ar e asymmetrical and are separated by septa (Fig. 2-5F). Sensilla styloconica (ss) are pres ent in the apical portion of the proboscis, with each sensillum consisting of a stylus with longitudinal ridges and an apical sensory cone (sc); proximal and apical regions ar e without visible sensilla trichoidea or cuticular processes. The ligulae (l) of the dorsal galeal crosslinkage are tria ngluar, and are restricted to the middle portion of the galea (Fig. 2-5E). The proboscides of selected spec ies from genera placed in the Primary piercers of thick-skinned fruit but secondary piercers of hard-skinned fruit: Anomis mesogona. The 44

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surface of the proximal region of the proboscis is simple and sm ooth with circular or semicircular ribs (Fig. 2-6A); depressions and cuticular processes ar e absent. Sensilla styloconica are modified into dorsoventrally flattened, ovate erectile barbs (eb), each with two lateral prominences and a triangular distal connus (Fi g. 2-6A). The erectile barbs are singular and sparsely positioned along the lateral side of the proximal galea (Fig. 2-6A). Erectile barbs are typically more abundant towards the apical region of the proboscis and are arranged on both lateral and ventrolateral sides (Fig. 2-6B). The apical regi on is nodulose with asymmetrical nodules throughout (Fig. 2-6B). The nodulose area at the tip of the proboscis is without septa (Fig. 2-6B). Furcate sensilla (fs) are also present in the apical portion of the proboscis, with each sensillum consisting of short and long triangular branches, some secondarily bifurcated. Sensilla basiconica are present only in apical region; pr oximal and apical regions are without visible sensilla trichoidea, or cuticular processes. The ligulae (l) of the dorsal galeal crosslinkage (dgl) are triangular (Fig. 2-6A). Calyptra canadensis. The surface of the pr oximal region of the proboscis is simple and smooth w ith circular or semi-circular ribs (Figs. 2-6C and 2-6D); depressions and cuticular processes are absent Sensilla styloconica are modified into dorsoventrally flattened, ovate erect ile barbs (eb), with a distinct distal connus (Figs. 2-6C and 26D). Individual erectile barbs are positioned in a single row along the lateral side of the proboscis and are positioned on vent rolateral sides in the apical region (Fig. 2-6C). Furcate sensilla (fs) are present along the lateral margin of the dorsal gale al crosslinkage (Fig. 2-6E). The furcate sensilla are four-pronged and asy mmetrical, consisting of two short and two long prongs, with a large sensory cone set in between (Fig. 2-6E). Th e surface of the apical region is smooth and the tip is with tear-shaped, socketed tearing hooks (Fig. 2-6D). Sensilla basiconica are positioned throughout the tip of the proboscis, typically arranged in groups of two or three 45

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(Fig. 2-6D). Proximal and apical regions are with out visible sensilla tric hoidea. The ligulae (l) of the dorsal galeal crosslinkage (dgl) are triangular (Fig. 2-6D). Calyptra lata. The surface of the proximal region of the proboscis is simple a nd smooth with circular or semi-circular ribs; depressions and cuticular processes are absent (F ig. 2-6F). Rasping sp ines (rs) are sparsely positioned along the lateral margin of the dorsal ga leal crosslinkage and on the ventrolateral sides of the proboscis and are without a distal connus (Fig. 2-6F ). The surface of the apical region is smooth and the tip is with tear-shape d, socketed tearing hooks (Fig. 2-6F). Sensilla basiconica (sb) are positioned thr oughout the tip of the proboscis, ty pically arranged in groups of two or three (Fig. 2-6F). Proxima l and apical regions are without visible sensilla trichoidea. The ligulae (l) of the dorsal galeal crosslinka ge (dgl) are triangular (Fig. 2-6F). Gonodonta nutrix. The surface of the proximal region of the proboscis is simple and smooth with circular or semicircular ribs; depressions are ab sent. Cuticular processes are ab sent from the ribs. Sensilla styloconica are modified into dorsoventrally flattened, ovate erectile barbs (eb), without a distinct distal connus (Fig. 2-7A ). Individual erectile barbs are positioned in a single row along the lateral side of the proboscis and are abundant in the apical region. Rasping spines (rs) are present along the lateral margin of the dorsal gale al crosslinkage (Figs. 2-7A and 2-7B). The surface of the apical region is smooth and the ti p is has cone-shaped, socketed tearing hooks (th, Fig. 2-7B); furcate sensilla are absent. Sensill a basiconica (sb) are posi tioned throughout the tip of the proboscis, typically arranged in groups of two or three (Fig. 2-7B). Proximal and apical regions are without visible sensil la trichoidea. The ligulae (l) of the dorsal galeal crosslinkage (dgl) are short and conical (Fig. 2-7B). Oraesia rectistria. The surface of the proximal region of the proboscis is simple and smooth with circular or semi-circular ribs; depressions and cuticular processes are absent. Sensilla styloconica are modified into dorsovent rally flattened, ovate 46

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erectile barbs (eb), with a distal connus (Fig. 2-7C). Erect ile barbs are abundant in the apical region of the proboscis, along with distinct raspin g spine-like (rs) structures The rasping spines triangular and without a distal connus. The surf ace of the apical region is smooth and the tip has tear-shaped, socketed tearing hooks (th, Fig. 2-7D). Furcate sens illa (fs) are present along the lateral margin of the dorsal galeal crossli nkage (Fig. 2-7C). The furcate sensilla are asymmetrical, consisting of one s hort and one long lateral prominen ce, with a large sensory cone set in between (Fig. 2-7C). Sensilla basi conica are positioned thr oughout the tip of the proboscis, typically arranged in groups of two or three (Fig. 2-7D ). Proximal and apical regions are without visible sensi lla trichoidea. The ligulae (l) of the dorsal galeal crosslinkage (dgl) are flattened and triangular (Fig. 2-7D). Plusiodonta compressipalpus. The surface of the proximal region of the proboscis is simple and smooth with circular or semi-circular ribs (Fig. 2-7E); depressions are absent. Cuticular processes or chaetiform sensilla (cs) are present at the base (Fig. 2-7E). Sensilla styloconica are modified into dorsoventrally flattened, ovate erectile barbs (eb), occasionally with a distin ct distal connus (Fig. 2-7E). Individual erectile barbs are positioned in a single row along the lateral side of the proboscis, and are more abundant in the apical region of the proboscis (Figs. 2-7E and 27F). Rasping spines (rs) are sparsely positioned along the lateral margin of the dorsal galeal cross linkage and are without a distal connus (Fig. 27F). The surface of the apical region is smoot h and the tip has tear-shaped, socketed tearing hooks (Fig. 2-7F). Sensilla basi conica (sb) are positioned thr oughout the tip of the proboscis, typically arranged in groups of tw o or three (Fig. 2-7F). Proximal and apical regions are without visible sensilla trichoidea. The ligulae (l) of the dorsal galeal cr osslinkage (dgl) are forked and triangular (Fig. 2-7F). Scoliopteryx libatrix. The surface of the proximal region of the proboscis is simple and smooth with circular or semi-circu lar ribs (Fig. 2-8A); depressions and cuticular 47

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processes are absent. Sensilla styloconica are modified into dorsovent rally flattened, ovate erectile barbs (eb) with two late ral prominences and a triangular di stal connus (Fig. 2-8A). The erectile barbs of the proximal galea are singular, and sparsely positioned al ong the lateral side of the proboscis (Fig. 2-8A). Erectile barbs are abu ndant in the apical regi on of the proboscis and are arranged on both lateral and ventrolateral sides (Fig. 2-8B). Th e surface of the apical region is heterogeneous with both nodulose and smooth regions (Fig. 2-8B). The nodulose area at the tip of the proboscis consists of areas with and without septa (Fig. 2-8B). The tip of the proboscis is smooth with a thin, band of cu ticle with slight ri dges extending into the nodulose portion (Fig. 2-8B). Sensilla basiconi ca are situated beneath the ridges at th e tip of the proboscis (Fig. 2-8B). Furcate sensilla (fs) are presen t in the apical portion of the proboscis, with each sensillum consisting of short and long triangular branches, so me secondarily bifurcated with setose distal processes. Proximal and apical regions are without visible sensilla trichoidea. The ligulae of the dorsal galeal crosslinkage are th in and triangular (Fig. 2-8A). Primary piercer of hard-skinned fruit: Eudocima homaena. The ventral surface of the proximal region of the proboscis is simple and sm ooth with diagonal circular or semi-circular ribs; depressions are absent. Chaetiform sensilla and other cuticular processes are not visible or are absent from the ribs. Two or three dorsoventrally flattened, triangul ar rasping spines (rs) occur just below the junction of the ribbed and sm ooth area of the apical region (Figs. 2-9A and 2-9B). Erectile barbs occur just below the juncti on of the ribbed and smooth areas of the apical region and are without a distinct distal connus (F ig. 2-9A and 2-9B). The surface of the apical region is smooth with serrated ridges (sr); the tip is cone-shaped, with socketed tearing hooks (th, Fig. 2-9A). Rasping spines and furcate sensilla are absent. Sensilla basiconica are positioned throughout the tip of the proboscis and on the tearing hooks, typically arranged in groups of two 48

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or three (Fig. 2-9B). Proximal and apical regi ons are without visible se nsilla trichoidea. The ligulae (l) of the dorsal galeal crosslinkage (dgl) are long and spike-like (Fig. 2-9B). The proboscides of selected Calyptra spp. in the Mammalian skin-piercers and bloodsucking : Calyptra eustrigata. The surface of the proximal region of the proboscis is simple and smooth with circular or semi-circular ribs (Fig s. 2-10A and 2-10B); depressions and cuticular processes are absent. Sensilla styloconica are modified into dorsovent rally flattened, ovate erectile barbs (eb), with a distinct distal connus (Figs. 2-10A ). Individual erectile barbs are positioned in a single row along the lateral side of the proboscis and are positioned on ventrolateral sides in the apical region (Fig. 2-10A). Furcate sensilla (fs) are present along the lateral margin of the dorsal galeal crosslinka ge (Fig. 2-10B). The furcate sensilla are symmetrical, consisting of two long lateral prom inences, with a longer sensory cone set in between (Fig. 2-10B). The surface of the apical re gion is smooth and the tip is with tear-shaped, socketed tearing hooks (th) (Figs. 2-10A-C). Sensilla basiconica (sb) are positioned throughout the tip of the proboscis, typically arranged in groups of two or three (Fig. 2-10B). Proximal and apical regions are without visible sensilla trichoidea. The ligulae (l) of the dorsal galeal crosslinkage (dgl) are triangular (Fig. 2-10B). Calyptra thalictri. The surface of the proximal region of the proboscis is simple and smooth with circular or semi-circular ribs (Figs. 2-11A); depressions and cuticular processes are absent Sensilla styloconica are modified into dorsoventrally flattened, ovate erect ile barbs (eb), with a distinct distal connus (Figs. 2-11A). The surface of the apical region is smooth and the tip is with sharp tear-shaped, socketed tearing hooks (th) (Figs. 2-11B and 2-11C). Proximal a nd apical regions are without visible sensilla trichoidea. The ligulae (l) of the dorsal galeal crosslinkage (dgl) are triangular (Fig. 2-11B). 49

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The proboscis of Tear drinking: Hemiceratoides hieroglyphica The placement of this species in this group at present is tentavive be cause nothing certain is known about its feeding habits. The most likely assumption is that it may pierce fruit and occasionally suck tears. The surface of the proximal region of the proboscis is simple and smooth with diagonal semicircular ribs (Fig. 2-12A). The ribs of the proximal regi on terminate into lateral plates with shallow cuticular depressions (Fig. 2-12D). Furcate sensilla (fs) are pres ent in the proximal and apical regions and occur along the lateral margin of the dorsal galeal crosslinkage (Figs. 2-12A, 2-12B, and 2-12D). The furcate sensilla are of two types (fs1 and fs2) both seemingly symmetrical: one consisting of a long, thin, feather-like plate with three small lateral prominences at the base, and the other with four prongs (two short and two lo ng) (Fig. 2-12D). Smooth sensilla styloconica (subtype 1, sss 1 ) are present in the apical region and are feather-like (Figs. 2-12A, 2-12B, and 212D). The apical region is with fixed, deltoid -pyramidal hooks (h), w ithout a distal connus (Figs. 2-12A-C). The surface of the apical region is smooth and the tip is with sclerotized ridges (sr) (Fig. 2-12C). Sensilla basiconica (sb) ar e positioned throughout the length of the galea (g), typically arranged in the center of the plates along the lateral margins of the dorsal galeal crosslinkage (Fig. 2-12D). Proximal and apical re gions are without visible sensilla trichoidea. The ligulae (l) of the dorsal galeal crosslinkage (dgl) are triangular (Fig. 2-12D). Uncertain Taxa : Phyllodes consobrinia The surface of the proximal region (pr) of the proboscis is simple and smooth w ith diagonal circular or semi-c ircular ribs; depressions and cuticular processes are absent. Sensilla styloconica are modified into thin, highy flattened, tearshaped erectile barbs (sensilla styloconica subtype 1, eb; Fig. 2-13A ), without a distal connus. Other sensilla styloconica are modified into a smooth, flattened recta ngular shape (subtype 2, sss 2 ), are abundant in the apical region of the proboscis, and are overlain along the lateral margin 50

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of the galea (Fig. 2-13A). The surface of the apical region is smoot h and the tip is with socketed tearing hooks and compressed furcate sensilla (fs) with asymmetrical edges. Sensilla basiconica are positioned throughout the tip of the proboscis and are located in the center of the smooth sensilla styloconica (subtype 2, sss 2 ), furcated sensilla, and tearing hooks (Figs. 2-13A and 213B). Proximal and apical regions are without visi ble sensilla trichoidea. The ligulae (l) of the dorsal galeal crosslinkage (dgl) are thin and tria ngular (Fig. 2-13A). Discussion This survey indicates it is likely the tearing hooks of the proboscis are restricted to the Calpini (sensu Zaspel and Branham 2008) and proboscis morphology is not strictly correlated, but is associated with feeding behavior (fruit piercing, blood feeding, or tear feeding). This study confirms that species with in the Calpini are equipped w ith piercing mouthparts and use them to pierce fruits wither as primary or s econdary piercers (Bnziger 1982). However, the reverse is not true for other pier cing species distantly related to members of Calpini. Bnziger (1982) had already noted that mout h-part structure alone is not sufficiently indicative of what fruit type a moth can pierce, but that it neve rtheless provides important clues: a thin, long, unsclerotized proboscis lacking pi ercing armature cannot conceivabl y, and indeed has never been seen to, penetrate the sound skin of longan. Also, while in Calyptra there are both hematophagous and typical fr uit-piercing species (e.g. C. lata C. hokkaida ), their proboscides are essentially the same and, despite minor diffe rences in levels of sclerotization, size and number of tearing hooks and erectile barbs characteristic for certain species, they are not predictive of their piercing cap ability or feeding habits (Bn ziger 1986, 2007). This is not entirely unexpected since hematophagous Calyptra are only facultatively so but at the same time obligatory fruit-piercers. Both light and scanning electron microscopy can be used to differentiate most of the charac ters of the proboscis. For example, the presence of tearing hooks, 51

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rasping spines, and shape of dor sal ligulae can be easily visualized using light microscopy. The surface microstructure, minor structural differen ces in the shape if the tearing hooks, and the presence of furcated sensillia are difficult to de tect without the availabi lity of scanning electron micrographs. However, the endocuticula that join s the tearing hooks with the sclerotized socket and also surrounds the erectile ba rbs are not visible by SEM but ar e evident by light microscopy. Specialized structures for blood feeding were not found in hematophagous Calyptra species. It is surprising that Bttiker et al. (1996) found significan t morphological adaptations in the proboscis of tear drinking moths. Despite th eir strongly modified behavior, tear feeders essentially imbibe fluid from a pool not unlike puddling Lepidoptera, for which no morphological adaptation is required. Since the purported adaptations to tear feeding are also found in females of species where only ma les are lachryphagous, and because of other reservations (Bn ziger 2007), the proboscis morphology in tear drinkers needs reassessment. A special case is H. hieroglyphica the biology of which is unknow n except for photographs, and description of observed movements of its probos cis inserted between th e eyelids of sleeping birds in Madagascar (Hilgartner et al. 2007; also see a reinte rpretation by Bnziger 2007). Scanning electron micrographs (SEM) of its pr oboscis clearly depict s numerous strong differences in microstructure when compared to those the SEMs of t ypical the tear-feeding moths (Bttiker et al. 1996; this report Figs. 2-11, 2-12); the armatu re is similar to that found in piercing proboscides. Further, tear-feeding H. heiroglyphica possess modifications of the proboscis that are similar to thos e found in fruit-piercing moths; however, it differs from Calpini proboscides in that the sensilla basiconica modified into hooks are fused to the proboscis, hence not movable by blood pressure, and in that the se nsilla styloconica which are modified into fixed erectile barbs are not movable by bl ood pressure. The proboscis of Eudocima spp. is different 52

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53 from that of Calyptra (including Oraesia Plusiodonta Gonodonta) in that it is larger and is the ventrally serrated. This ventral serration, plus the st ronger, more robust proboscis, is the main reason why Eudocima is able to pierce hard skin ned fruit (longan, litchi) while Calyptra and the other genera are not. Some piercing noctuid taxa not in cluded in the survey such as Ercheia spp., Pericyma spp., Serrodes spp. have proboscis armatures that are in some respects similar to Calyptra and other calpines, while others (e.g., Facidina suffumata, Platyja spp., Saroba albopunctata and Acherontia spp.) lack normal armature (teeth, hooks, barbs, serrati ons, ridges) altogether but pierce with a pointed, fully sclerotized terminal sect ion of the proboscis s uggesting that fruitpiercing has evolved multiple times within the Noct uidae. The glossary of proboscis structures in this study provides criteria for which homologies can be assessed across a broad range of taxa; the exact function(s) of some structures still needs to be tested (e.g., furcate sensilla), but proposed function based on the prim ary literature is mentioned. I conclude that proboscis morphology needs careful examination in its application for reconstructing a natural classification of piercing moths and predicting of observed differences in adult feeding behavior. The above-mentioned armature of tearing hooks and er ectile barbs is charac teristic for the tribe Calpini and thus should be re stricted to its members rath er than the subfamily.

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Table 2-1. Sensilla and other structures associated with calpine proboscides and their proposed function Structure ( Abbreviation ) Definition/Criterion *reference s for terminology are listed in methods section Proposed function Cuticular Hooks (h) : Fixed, deltoid-pyramid to wedge-like structures. Unknown. Dorsal Galeal Ligulae (dgl) : Zipper-like structure comprise d of glossae and paraglossae. Holds tubes of the proboscis together. Erectile Barbs Subtype 1 (eb) : Modified sensilla styloconi ca, apical connus present, surr ounded by endocuticular material. Mechanoreception; possibly contact chemoreception. Ligula (l) : Individual zipper-like structures comprised of glossae and paraglossae. Form dorsal and ventral galeal crosslinkages of proboscis. Smooth Sensilla Styloconica Subtype 1 (sss 1 ) : Flattened, feather-like w ith a basiconic sensillum but without distal connus. Contact chemo-mechanoreceptors. Smooth Sensilla Styloconica Subtype 2 (sss 2 ) : Flattened, rectangular with a basico nic sensillum but without distal connus. Contact chemo-mechanoreceptors. Furcated Sensilla (fs): Cuticular styloconic sensilla, of ten assymetrical, branched with hairs or finger-like projections. Unknown, but may perceive mechanical distortions. 54 Rasping Spines (rs): Finger-like, flattened, aporous structur es, without hairs or a sensory cone. Structural, possibly contact chemorecpetors. Sensilla Basiconica (sb) : Peg-shaped sensilla with minute pores. Olfactory structures, chemoreceptors. Sensilla Styloconica (ss) : Cuticular structures consisting of a ba siconic peg elevated on a style or cone ( sc ) Contact chemo-mechanoreceptors. Sensilla Trichodea (st) : Cuticular, hairlike projection, aporous. Mechanoreceptors, function in food localization. Serrated Ridge (sr) : Ventrally serrated cuticul ar ridge located on vent ral side of proboscis. Structural, used for piercing hard -skinned fruit (e.g., longan, litchi). Tearing Hooks (th) : Aporous, cuticular structure with a collar, moveable by blood pressure, with or without a basiconic sensillum, attached to socket via elastic endocuticula. Structural, involved in piercing through fruit or mammalian ti ssue, possible mechanoreception. Minute Triangular Spines (mts) : Cuticular structures, membraneous. Possibly used in brushing eye of host to induce production of tears.

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Table 2-2. Specimens examined A. Genus species author Feeding Group Collection Country of Origin SEM Light Microscope Anomis mesogona (Walker) PTS FLMNH Taiwan X X A. privata (Walker) PTS FLMNH Taiwan X X Calyptra albivirgata (Hampson) PTS NMNH China X C. bicolor (Moore) PTS/MSP NMNH Nepal X C. bicolor PTS/MSP NMNH Nepal X C. canadensis (Bethune) PTS AMNH USA X X C. eustrigata (Hampson) PTS/MSP NMNH Thailand X X C. eustrigata PTS/MSP NMNH Malaysia X C. fasciata (Moore) PTS/MSP Fibiger Nepal X C. fletcheri (Berio) PTS/MSP* NMNH Nepal X C. gruesa (Draudt) PTS NMNH China X C. lata (Butler) PTS FLMNH Slovakia X X C. lata PTS NMMH S. Korea X X C. minuticornis (Guene) PTS/MSP NMNH Thailand X 55 C. minuticornis PTS/MSP Fibiger Nepal X C. ophideroides (Guene) PTS/MSP NMNH Himalaya X C. ophideroides PTS/MSP NMNH India X C. orthograpta (Butler) PTS/MSP NMNH China X C. orthograpta PTS/MSP NMNH Thailand X C. parva Bnziger PTS/MSP NMNH Thailand X C. pseudobicolor Bnziger PTS/MSP NMNH Nepal X C. pseudobicolor PTS/MSP NMNH Nepal X C. subnubila (Prout) PTS NMNH Indonesia X C. thalictri (Borkhausen) PTS/MSP NMNM Austria X X C. thalictri PTS/MSP* Fibiger Russia (RFE) X X ______________________________________________________________________________________________________

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Table 2-2. Continued Genus species author Feeding Group Collection Country SEM Light Microscopy Image Eudocima homaena (Hbner) PHS Fibiger Indonesia X X Eudocima salaminia (Cramer) PHS NMNH Papua New Guinea X Ferenta castula (Dognin) UT NHM Colombia X Goniapteryx servia (Stoll) NP FLMNH USA X X Gonodonta nutrix (Cramer) PTS USNM Brazil X X Graphigona regina (Guene) UT NHM Costa Rica X Hemiceratoides hieroglyphica (Saalmller) TD NMNH Malawi X X Hypsoropha hormos Hbner NP FLMNH USA X X Oraesia argyrosigna Moore PTS NMNH Taiwan X O. argyrosigna PTS NMNH Tanzania X O. argyrosigna PTS Fibiger Nepal X O. argyrosigna PTS Fibiger Nepal X O. emarginata (Fabricius) PTS NMNH Malaysia X O. emarginata PTS NMNH Sri Lanka X O. excavata (Butler) PTS NMNH Japan X 56 O. excitans Walker PTS NMNH Mexico X O. glaucochelia (Hampson) PTS NMNH Bolivia X O. honesta Walker PTS NMNH Mexico X O. honesta PTS NMNH Mexico X O. nobilis Felder and Rogenhofer PTS NMNH Brazil X O. provocans Walker PTS NMNH Malawi X O. rectistria Guene PTS FLMNH India X O. rectistria PTS NMNH Nepal X X O. serpans Schaus PTS NMNH Venezuela X O. serpans PTS NMNH Venezuela X O. striolata Schaus PTS NMNH Peru X O. striolata PTS NMNH Bolivia X _______________________________________________________________________________________________________

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57 Table 2-2. Continued Genus species author Feeding Group Collection Country SEM Light Microscopy Image O. triobliqua (Saalmller) PPTS NMNH Malawi X O. triobliqua PTS NMNH Rhodesia X O. wintgensi (Strand) PTS NMNH Unknown X Phyllodes consobrina Westwood PTS FLMNH Assam X X Phyprosopus callitrichoides Grote NP FLMNH USA X X Plusiodonta coelonota (Kollar) PTS NMNH USA X X Plusiodonta compressipalpus Guene PTS FLMNH USA X X Plusiodonta incitans (Walker) PTS NMNH Mexico X Plusiodonta incitans PTS NMNH Argentina X Scoliopteryx libatrix (L.) PTS Fibiger Denmark X X Scoliopteryx libatrix PTS FLMNH USA X Tetrisia florigera Walker UT NHM Peru X ____________________________________________________________________________________________________________

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Figure 2-1. Feeding behaviors of adult moths in the subfamily Calpinae. (A) Oraesia rectistria Guene piercing plum in Nepal (photo J.M. Zaspel), (B) Anomis fructusterebrans Bnziger piercing yellow Himalayan raspberry ( Rubus ellipticus Sm.) in N. Thailand (photo H. Bnziger), (C) Scoliopteryx libatrix (L.) piercing raspberry in Switzerland (photo H. Bnziger), (D) Eudocima tyrannus Guene piercing apple in Korea (photo H. Fay), (E) Hemiceratoides hierglyphica feeding on the tears of a bird in Madagascar (photo R.D. Hilgartner), (F) Calyptra thalictri feeding on blood from human thumb in Far Eastern Russia (photo J.M. Zaspel). 58

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Figure 2-2. Description of proboscis regions; Oraesia rectistria 59

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Figure 2-3. Examples of proboscis structures visible by light mi croscopy in selected feeding categories. Primary piercers of thick-ski nned fruit but secondary piercers of hardskinned fruit (A), Oraesia serpans ; Proximal region, dgl = dorsal galeal ligulae, eb = erectile barbs subtype 1, (B) O. serpans ; Apical region, th = tearing hooks, (C) Gonodonta nutrix ; Proximal region, dgl = dorsal galeal ligulae, eb = erectile barbs subtype 1. Primary piercers of hard-skinned fruit, (D) Eudocima homanea; Proximal region, dgl = dorsal galeal ligulae, rs = rasp ing spines; Apical region, sr = serrated ridges, th = tearing hooks. Mammalia n skin piercing and blood feeding, (E) Calyptra fasciata ; Proximal region, dgl = dorsal galeal ligulae, eb = erectile barbs subtype 1, (F) Calyptra fasciata ; Apical region, th = tearing hooks. 60

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Figure 2-4. Proboscis of non-calpine classical tear feeder (A) Lobocraspis griseifusa ; Apical region 1, dgl = dorsal galeal ligulae, ss = sensilla styloconica, (B) L. griseifusa Apical region, ss = sensilla styloconica. 61

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Figure 2-5. Proboscides of taxa in th e non-piercing fruit-sucking group. (A) Goniapteryx servia ; Proximal region, dgl = dorsal galeal ligulae, Ap ical region 1, ss = se nsilla styloconica, (B) G. servia ; Apical region 2, l = ligula, ss = sens illa styloconica, sc = sensory cone, (C) Hypsoropha hormos ; Apical region, ss = sensilla styloconica, (D) H. hormos ; Proximal region, dgl = dorsal galeal ligulae, g = galea, l = ligula, ss = sensilla styloconica, sc = sensory cone, (E) P. callitrichoides ; Proximal region, l = ligula, (F) P. callitrichoides ; Apical region, ss = sensilla st yloconica, sc = sensory cone. 62

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Figure 2-6. Examples of probosci s structures visible by scanni ng light microscopy in primary piercers of thick-skinned fruit but secondary piercers of hard-skinned fruit. (A) Anomis mesogona; Apical region 1, eb = erectile barb s, fs = furcated senislla, sc = sensory cone. (B) A. mesogona; Apical region 2, eb = erectile barbs, fs = furcated senislla, sc = sensory cone. 63

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Figure 2-6 (continued.) Examples of proboscis structures visible by scanning light microscopy in primary piercers of thick-skinned fruit but s econdary piercers of hard-skinned fruit. (C) Calyptra canadensis ; Apical region 1, eb = erectile ba rbs, fs = furcated sensilla, l = ligula, th = tearing hooks, (D) C. canadensis ; Apical region 2, sb = sensilla basiconica, sensory cone, (E) C. canadensis ; Apical region 3, fs = furcated sensilla, (F) C. lata ; Proximal region, rs = rasping spin es, Apical region, sb = sensilla basiconica, th = tearing hooks. 64

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Figure 2-6 (continued.) Examples of proboscis structures visible by scanning light microscopy in primary piercers of thick-skinned fruit but s econdary piercers of hard-skinned fruit. (F) Calyptra lata ; Proximal region, rs = rasping spin es, Apical region, sb = sensilla basiconica, th = tearing hooks. 65

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Figure 2-7. Examples of probosci s structures visible by scanni ng light microscopy in primary piercers of thick-skinned fruit but seconda ry piercers of hard-skinned fruit II. (A) Gonodonta nutrix ; Proximal region, g = galea, Apical region 1, eb = erectile barbs, rs = rasping spines, th = tearing hooks, (B) G. indentata ; Apical region 2, eb = erectile barbs, l = ligula, rs = rasping spines, sb = sensilla basiconica, th = tearing hooks, (C) Oraesia rectistria ; Apical region 1, eb = erectile ba rbs, fs = furcated sensilla, l = ligula, sb = sensilla basiconica, rs = rasping spines, th = tearing hooks, (D) O. rectistria ; Apical region 2, l = ligula, sb = sens illa basiconica, th = tearing hooks. 66

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Figure 2-7. Continued 67

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Figure 2-8. Examples of probosci s structures visible by scanni ng light microscopy in primary piercers of thick-skinned fruit but seconda ry piercers of hard-skinned fruit III. (A) Scoliopteryx libatrix ; Proximal region, eb = erectile barbs, sc = sensory cone, Apical region 1, sb = sensilla basiconica, (B) S. libatrix ; Apical region 2, eb = erectile barbs, fs = furcated sensilla, sc = sensory cone. 68

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Figure 2-9. Proboscis of taxa in the primar y piercing of hard-ski nned fruits group. (A) Eudocima homaena; Apical region 1, dgl = dorsa l galeal ligulae, g = galea, sr = serrated ridges, (B) E. homaena; Apical region 2, l =ligula, rs = rasping spines, sb = sensilla basiconica, sr = serrated ridges, th = tearing hooks. 69

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Figure 2-10. Proboscides of taxa in the mamma lian skin-piercing and blood-feeding group. (A) Calyptra eustrigata ; Apical region 1, eb = erectile ba rbs, sb = sensilla basiconica, sc = sensory cone, th = tearing hooks, (B) C. eustrigata; Apical region 2, fs = furcated sensilla, sb = sensilla basiconica, (C) C. eustrigata ; Apical region 3, th = tearing hooks. 70

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Figure 2-11. Proboscides of taxa in the mammalian skin-piercing and bloo d-feeding group II. (A) C. thalictri ; Apical region 1, eb = erectile barbs, fs = furcated sensilla, sc = sensory cone, (B) C. thalictri ; Apical region 2, l = ligul a, th = tearing hooks, (C) C. thalictri ; Apical region 3, th = tearing hooks. 71

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Figure 2-12. Proboscides of taxa in the tear-drinking group. (A) Hemiceratoides hieroglyphica ; Proximal region, fs = furcated sensilla, g = galea, sb = sensilla basiconica, Apical region 1, sss 1 = smooth sensilla styloconica subtype 1, h = cuticular hook, (B) H. hieroglyphica ; Apical region 2, h = cuticular hoo k, sb = sensilla basiconica, sss 1 = smooth sensilla styloconica subtype 1, (C) H. hierglyphica ; Apical region 3, fs = furcated sensilla, h = cuticular hook, l = ligula, sb = sensilla basiconica, sr = serrated ridge, (D) H. hierglyphica ; Apical region, fs = furcated sensilla (1 and 2), l = ligula, sb = sensilla basiconica. 72

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Figure 2-13. Uncertain taxa. (A) Phyllodes consobrina ; Apical region 1, dgl = dorsal galeal ligulae, eb = erectile barbs, l = ligula, sb = sensilla basiconica, sss 2 = smooth sensilla styloconica subtype 2, th = tearing hooks, (B) Phyllodes consobrina ; Apical region 2, sb = sensilla basiconica, sss 2 = smooth sensilla styloconica subtype 2, th = tearing hooks. 73

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CHAPTER 3 RECONSTRUCTING THE EVOLUTIONARY RELATIONSHPS OF THE VAMPIRE MOTHS AND THEIR FRUITPIERCING RELATIVES USI NG MORPHOLOGICAL AND MOLECULAR DATA (LEPIDOPTERA: NOCTUIDAE: CALPINAE: CALPINI) Introduction Hematophagy is believed to have arisen independently in arthr opods during the Jurassic and Cretaceous periods at leas t six, and potentially as many as 21, times (Balashov 1984, Ribeiro 1995). Adams (1999) estimates that 14,000 insect species from five orders (Pthiraptera, Diptera, Hemiptera, Lepidoptera, and Siphonaptera) are hematophagous. Within Lepidoptera, skin piercing and blood feeding are re stricted to the moth genus Calyptra Ochsenheimer (Fig. 3-1). Calyptra includes what are commonly known as vampire moths, so named because of their ability to pierce mammalian skin and feed on blood. Calyptra spp. are medium-sized, with wingspans ranging from 36-72 mm. Species in this genus occur in Europe, eastern Africa, sub-Hi malayan regions of S. Asia, the Manchurian subregion, and are broadly dist ributed throughout S.E. Asia. Calyptra species have modified proboscides equipped with strongly sclerotized barbed hooks used for piercing the skin of hard fruits such as peaches and citrus, and occasio nally of mammals (Bnziger 1982, Zaspel et al. 2007, Zaspel 2008). Of the seventeen species described (Bnziger 1983), ten male Calyptra species have been observed piercing mammalia n skin and feeding on blood (Bnziger 1989, Zaspel et al. 2007). Males of these ten species are facultative blood feeders; females have not been documented feeding on blood. It is possibl e that the male moths may seek out mammalian hosts to obtain additional nutrients such as amino acids or sugars thereby increasing fitness, but the blood meal itself does not appear to incr ease longevity (Bnziger 2007). Blood-feeding Calyptra males have not tested positive for pr otesases, indicating amino acids are not sequestered; however, male moths do appear to be in search of salts (Bnziger 2007). It is 74

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possible that the males are seque stering salts and transferring th em to the females during mating for egg production (Smedley and Eisner 1995) or to replenish salt supplies depleted during oviposition (Adler and Pearson 1982). At least eight additional closely related genera ( Africalpe Eudocima Ferenta Gonodonta, Graphigona, Oraesia and Plusiodonta and Tetrisia ) have homologous proboscis modifications used for fruit-piercing, but the occurrence of blood-feedi ng in those species has not been observed (Bnziger pers. com., Zaspel 2007). Bnziger (1982) described categ ories of fruits based on thei r increasing difficulty to be pierced by moths, e.g., very soft-skinned fruit (e .g. raspberry), soft-skinned fruit (e.g. peach, grape), thick-skinned fruit (e.g. citrus), and hard-skinned fruit (longan, lichi). Moths were grouped according to their ability to pierce the four categories as primary piercers. Primary piercers are able to penetrate the skin of the fru it, while secondary fruit piercers are only capable of piercing fruit damaged previously by primary piercers or other animals (Bnziger 1982). For example, a moth like Calyptra minuticornis is a primary piercer of th ick-skinned fruits (oranges) and all softer-skinned fruit, but a secondary piercer of hard-skinned fruit (longan). The other feeding types, i.e. non-piercing fruit sucking, nectar sucking, non-piercing blood sucking, skin piercing blood sucking, and the va rious degrees of lachryphagy, were characterized in Bnziger (1973, 2007). Some feeding groups include taxa that exhibit polyt ypic feeding behaviors; thus, continuity or overlap between feeding types has been observed for some species. It has been hypothesized that an evolutionary progression from secondary to primary fruit piercing has culminated in skin piercing and blood feeding in these moths (Fig. 3-2: D, Bnziger 1971, 1989). Alternative hypotheses regard ing the evolution of feeding in Calyptra have been proposed (Downes 1973, Hilgartner et al. 2007) and suggest the skin-piercing and blood-feeding 75

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behavior is derived from other animal-associated feeding beha viors such as dung, urine, or tear feeding (Figs. 3-2: B and C, respectively). This evolutionary trajectory is an unlikely given the proboscis structures of fruit pier cers and blood feeders are not ho mologous with those of tearfeeding moths, and such lachr ophagous moths do not pierce fru it. This hypothesis is also problematic given the shared be havioral modifications found in both fruit piercing and blood feeding moths. The tearing struct ures involved in the piercing of fruits or mammalian skin is restricted to a small group of taxonomically a ssociated noctuid genera (Zaspel, unpublished data), while animal-associated feeding behaviors, including th e imbibing of blood droplets found on the bodies of mammals, are widespread w ithin Lepidoptera (Bnzi ger 1982, Scoble 1992). These feeding hypotheses have never been tested within an empirical phylogenetic framework, and a hypothesized directional progression of feed ing types cannot be test ed formally until the relationships of Calyptra and related genera are known. The primary purpose of this study is to reconstruct a phylogeny of Calpini to determine evolutionary relationships among the genera in Calpini and related tribes based on morphological and molecular characters. The evolution of feeding behaviors is also invest igated by using the resulting phyloge ny to test the hypothesis of a directional progression of feeding types from nectar feeding to fruit-piercing to skin-piercing and blood-feeding in these calpine moths. Materials and Methods Taxon Sampling The most recent classifications place Calp inae in the family Noctuidae (Kitching and Rawlins 1998, Fibiger and Lafontaine 2005, Lafontaine and Fibi ger 2006, Mitchell et al. 2006). Calpinae consists of four tribes: Anomini, Calpini, Phyllodini, and Scoliopterygini (Fibiger and Lafontaine 2005, Lafontaine and Fibiger 2006, Holloway 2005). All genera in Calpini contain fruit-piercing species (Fibig er and Lafontaine 2005, Holloway 2005, Zaspel and Branham 2008); 76

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a high concentration of economically important fru it-piercing species is f ound within the tribe. For the phylogenetic review of blood-feeding moths and related genera, 193 specimens were dissected, representing species from the following genera: Africalpe Eudocima Calyptra Ferenta Goniapteryx, Gonodonta, Graphigona, Hemiceratoides, Hypsoropha, Oraesia Phyprosopus Plusiodonta and Tetrisia Walker. Representatives from the three other tribes in the Subfamily Calpinae were also examined: Anomis flava and A. mesogona (Anomini), Phyllodes consobrina Westwood (Phyllodini), and Scoliopteryx libatrix (Scoliopterygini). The following is a list of the institutional and privat e collections consulted during this study. The acronym of the institution or name of private collection is followed by the name of the individual that prepared the loan. Acronyms follow Heppner and Lamas (1982): AMNH American Museum of Natural History, New York (T. Sc huh); MGCL McGuire Center for Lepidoptera and Biodiversity, Gainesville (G. Austin); HNM Hungarian Natural History Museum, Budapest (L. Ronkay); NHM Natural Hi story Museum, London (M. Honey); NMNH National Museum of Natural History, Washi ngton D.C. (M. Pogue); Queensland, (H. Fay); UMD University of Maryland (C. Mitter); MF Personal collection of Michael Fibiger; HB Personal collection of H. Bnziger, Chiang Mai, Thailand (HB has donated the material used in this study to the NMNH). Taxa included in morphological phylogenetic analyses. Ingroup taxa were selected based on checklists of th e tribe Calpini (Fibiger and Laf ontaine 2005, Lafontaine and Fibiger 2006, Holloway 2005, Zaspel and Branham 2008), generic checklists (Poole 1989, Zilli and Hogenes 2002), and previous species and gene ric associations publis hed by other authors (Hampson, 1926, Bnziger 1983, Hilgartner et al. 2007). Taxa were also selected based on the availability of material, includ ing the availability of male-female pairs; type species for all 77

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genera in the analysis were also examin ed and included. Sixteen of eighteen of Calyptra twelve of forty-six species of Eudocima ten of forty-one Gonodonta, one of one described Graphigona species, both described species of Hemiceratoides two of four described Hypsoropha species, eleven of twenty-four Oraesia species, one of nine Phyprosopus species, and seven of thirtyseven described Plusiodonta species were included in the study. The following taxa were used as outgroups: Anomis mesogona, A. flava and Scoliopteryx libatrix Taxa included in molecular phylogenetic analyses. DNA was extracted from as many species from the morphological dataset as possible. Because many of the taxa studied occur in remote areas, fresh specimens were not readily available. Museum material was used when permission was given and if the material was le ss than 30 years old. Taxa included in the molecular dataset are listed in Table 3-1. Taxa omitted from phylogenetic analyses. A single female of Phyllodes consobrina was examined but was not included in the analysis b ecause it lacked clear similarities to the other study genera. The placement of this genus is pr oblematic as it shares characteristics with both Calpini and Ophusini (Holloway 2005). Hollowa y (2005) assigns this genus to the tribe Phyllodini; it is possible th at the exact placement of Phyllodes in this tribe needs further assessment. Goniapteryx servia males and females were also ex amined but were also excluded from all analyses because they were too dive rgent with respect to other study genera and homology statements could not be made with confidence. Complete male-female pairs of Africalpe Ferentia and Tetrisia were unavailable at the time of the study. Morphological Data Dissection methodology follows Winter (2000) and is fully described in Zaspel and Weller (2006). Most wings were not clea red and slide-mounted. Euparol mounts were 78

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transferred from the dehydration series into a final 15-minute treatment in Euparol essence (Bioquip, Garden City, CA) before slide mounting. Permanent slide mounts (Canada balsam [Sigma, St Louis, MO] or Euparo l [Bioquip, Garden City, CA]) we re made of abdominal pelts, genitalia, legs, wings, labi al palps and antennae. Slides were placed on trays and cured in drying ovens for 24 48 hours. Terminology. Terms for abdominal and genitalic morphology follow Klots (1970), Bnziger (1983), Fo rbes (1960), Weller et al. (2000) Jacobson and Weller (2002), Goater et al. (2003), and Kriste nsen (2003). Terms for probosci s morphology follows Zaspel et al. (submitted). A set of morphological characters based on previous studies (Bnziger 1983, Jacobson and Weller 2002, Goater et al. 2003, Holloway 2005, Zaspel and Weller 2006) was compiled and included characters from the head, appendages, male and female genitalia. Thirteen new characters were described from the proboscis and ar e figured in Chapter 2. A total of sixty-six characters and two hundred nine ty-nine character states were coded. A morphological data matrix with with twenty-six binary characte rs and forty unordered multistate characters was scored. Inapplicable character states were coded as mi ssing (?) (Strong and Lipscomb 1999). Characters exhibiting intr aspecific variation were not code d. The inclusion of the proboscis characters in the analysis in order to examine th e evolution of piercing be haviors is controversial (Coddington 1988, McLennan et al. 1988); their presence in the data ma trix may lead to a lack of independence between the actua l morphological characters an d ecological hypotheses thus biasing the analyses (Luckow and Bruneau 1997, de Queiroz 1996). However, Luckow and Bruneau (1997) state, charact er exclusion can lead to a w eaker phylogenetic hypothesis, and also, characters as statemen ts of homology differ from characters as statements of functionality. Thus, the characters of the pr oboscis were coded as statements of homology and 79

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included in the analysis, and th e feeding behaviors as statemen ts of functionality and were excluded from the analysis and mapped onto the resulting phylogeny. Additionally, the proboscis structures can be c onsidered independent from th e feeding behaviors because probability of one type of piercing behavior (e.g., fruit-piercing or skin-piericng and bloodfeeding) is not necessarily correlated with particular proboscis structures. The final morphological data matrix include d sixty-five taxa, fi fty-seven of which were complete malefemale pairs, five species were represented by females only, and three were represented by males only. Molecular Data Available fresh tissues for as many moth species as possible from the morphological dataset were stored at -80 C. DNA extractions from fresh moth legs and dried, pinned material (10-30 years old than 20 years old) were performed using th e DNeasy Tissue Extraction Kit (Qiagen) and PUREGENE reagents (Gentra Sy stems, Minneapolis, MN). Samples were centrifuged for 15 min at 12,000 rpm and the supernatant was removed and transferred into a clean 1.5-mL centrifuge tube. DNA was precipitate d using isopropanol at -80C. Samples were centrifuged for 15 min at 12,000 rpm and the DNA pellets were washed with 70% EtOH, air dried for 5 min, and re-suspended in 100-L of sterile water. In or der to prevent contamination of the surface-sterilized sample s, all DNA extractions were performed in an area separated from where the high-fidelity PCR was conducted; voucher labels were assigned a nd placed in vials or on pins with the remaining moth bodies (Table 3-1). A 50-L high-fidelity PCR kit (Bioline, Randolph, MA) was used to amplify 1-L of template DNA with the following PCR reaction condi tions and with the pr imers listed in Table 3-2. Agarose gel electrophoresis (1% TAE gels) was used to separate PCR-amplified DNA, which was stained with ethidium bromide and visu alized with ultraviolet light. Double stranded 80

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High-Fidelity PCR products were purified using a Qiagen PCR purification kit and sequenced directly at the ICBR Core facility at the Univ ersity of Florida. Se quences were downloaded, trimmed, and aligned using MUSCLE (Edgar 2004). The molecular data set includes nearly comp lete sequences for one segment near the 5 end of the cytochrome c oxidase I (COI) mitochondrial ge ne and a fragment of the nuclear large subunit (28S, D2 region) rRNA. Partial COI sequ ences were included for the following species: Calyptra albivirgata Eudocima salaminia, Hemiceratoides sittaca and Oraesia emarginata. The relevance of these regions for phylogeny in ference has been demonstrated in previous studies (Brower and Egan 1997, Br ower et al. 1997, Caterino et al. 2001, Hajibabaei et al. 2006, Weller et al. 1992, Mardulyn et al. 1999, Megens et al. 2004, Monteiro and Pierce 2001, Niehuis et al. 2006, Wahlberg and Zimmerman 2000). The two gene regions span 1427 bp in total: 665 bp, 696 bp, respectively. Due to a lack of suitabl e specimens, the molecular dataset does not include all species from th e morphological dataset. Phylogenetic Analyses Phylogenetic trees of separate and combined morphological and molecular data sets were constructed using parsimony anal yses implemented in TNT Version 1.0 (Goloboff et al. 2003). Bayesian analyses of molecular datasets were conducted using MRBayes 3.1 (Huelsenbeck and Ronquist 2001). Models were fit to molecula r data using the program MODELTEST (Posada and Crandall, 1998) and morphologi cal partitions using the pr ocedures described by Lewis (2001), Nylander et al. (2004), and Ronquist a nd Huelsenbeck (2005) allowing for comparison between maximum parsimony (MP) and Bayesian inference (BI) topologies. Resulting parsimony and Bayesian topologies were compar ed for overall similarity using procedures described by Nye et al. (2006). Branch suppor t for morphological data was calculated using 81

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jackknife resampling and combined datasets were calculated using nonparametric bootstrap resampling (Felsenstein 1985). Evolution of Feeding Behaviors and Complementary Analyses Comparative studies elucidate evolutionary trends by comparing certain characteristics, i.e. descriptions of the environments inhabi ted by the organisms, phenotypic characters, and behaviors across taxa represented in a phyl ogeny (Harvey and Pagel 1991). Comparative analyses of feeding behaviors re quire that those beha viors and hosts of th e fruit-piercing and blood-feeding moths for each taxon are known (Har vey and Pagel 1991). These data for the moth species under investigation have been summarized (Bnziger 1982, Bosch 1971, Fay 2002, Fay and Halfpapp 1999, Hargreaves 1934, Hatori 1962, Hilgartner et al. 2007, Huber et al. 1998, King and Thompson 1958, Maff 1990, Nomura and Hatori 1967, Reddy et al. 2007, Sands and Shotz 1991, Todd 1959, Whitehead and Rust 1972, Yoon and Lee 1974, Zaspel et al. 2007, Zaspel Chapter 2; Table 3-3). Observational da ta for many species in the tribe were extracted from available literature. In some cases, species were recorded as proba ble primary piercers or established primary piercer of various fruit type s. Also, observational data for some taxa have only been recorded under laboratory conditions as opposed to natural conditions. Lack of observational data for some species does not mean that the species does not pierce fruit or mammalian skin. Due to some incomplete feeding behavior data in the literature the following assumptions were made. When a report described a moths feeding behavior as possible or if a feeding behavior was unknown it was coded as abse nt (0). When a report described a moths feeding behavior under laboratory conditions it was treated as presence (1) of that particular feeding behavior. Because feeding behaviors are polytypic for many taxa under investigation (fruit-piercers also suck necta r), they were coded as present (1) for that feeding category. Feeding behaviors were divide d into the following functional feeding categories: A) Non82

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piercing fruit-sucking, B) Primary piercing of thick-skinned fruit, C) Secondary piercing of hardskinned fruit, D) Primary piercing of hard-ski nned fruit, E) Mammalian skin piercing and bloodfeeding, and F) Tear feeding. When known, binary feeding behavior char acters were coded for all taxa in the morphological matrix in MacC lade 4.0 (Maddison and Maddison 2000). Feeding behaviors for all taxa were mapped onto the resulting topology using parsimony optimizations (ACCTRAN, DELTRAN), and equi vocal cycling in MacClade version 4.08 (Maddison and Maddison 2000). Results and Discussion Summary of Morphological Character Variation The final data matrix included both non-genital and genital characters. The characters of the proboscis showed unexpected variation be tween ingroup and outgroup taxa. Significant differences in the surface micros tructure were observed between Hypsoropha and Phyprosopus spp. and other calpine genera. Hypsoropha and Phyprosopus have a fluted proximal proboscis region, while the other taxa included in the anal ysis have the simple and smooth condition. All outgroup genera were lacking the tearing hooks, erectile barbs, and rasping spines. These three characters are unreversed synapomorphies for the tr ibe Calpini. While there was some variation in proboscis structures between outgroup and i ngroup genera, the characters were typically similar within genera. The lack of variati on in proboscis characters among ingroup genera is surprising and suggests proboscis morphology is not tightly correlated with feeding type. All species within Gonodonta, Oraesia and Calyptra have identical probosci s armature yet bloodfeeding species have only been documented in the genus Calyptra In general, characters of the male aedeagus were highly variable be tween species and thus few characters were described from this system Other characters of the male and female genitalia were variable, but to a lesser degree, and thus were incl uded in the analysis. The shape 83

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of the male valve and characters of the labial palps were highly informative and diagnostic at the generic-level (e.g., Plusiodonta and Gonodonta, respectively). Other characters from the male genitalia were highly informative such as the pattern of sclerotization of sternite VIII (CI = 0.90, RI = 0.94). Informative characters from the female genitalia include the sh ape of the antevaginal plate (Fig. 3-11; CI = 0.57, RI = 0.67) and the sh ape of segment VIII (Fig. 3-12; CI = 0.74, RI = 0.84). All morphological characters, their states, consistency, and retention indices are described below. Head and appendage characters 01 Surface microstructure of proximal region of proboscis. 0, fluted; 1, simple and smooth (CI = 1.00; RI = 1.00). 02 Surface microstructure of proximal region of proboscis. 0, without circular ribs; 1, with circular ribs and longitudinal depressions; 2, with semicircular ribs but without longitudinal depre ssions (CI = 1.00; RI = 1.00). 03 Circular ribs with cuticular processes. 0, absent; 1, present (CI = 1.00; RI = 1.00). 04 Surface microstructure of apical region of proboscis. 0, densely nodulose; 1, smooth (CI = 1.00; RI = 1.00). 05 Apex of proboscis. 0, smooth; 1, nodulose; 2, serrate; 3, heterogeneous, both smooth and nodulose (CI = 1.00; RI = 1.00). 06 Nodules of near ligulae of the dorsal galeal cross linkage. ?, does not apply; 0, contiguous; 1, separated by septa (CI = 1.00; RI = 1.00). 07 Erectile barbs (eb) occurring along exte rior lateral margin of proboscis. 0 absent; 1, present (CI = 0.50; RI = 0.83). 08 Rasping spines (rs) occurring along lateral margin of proboscis. 0, absent; 1, present (CI = 1.00; RI = 1.00). 09 Furcate sensilla. 0, absent; 1, present, symmetrical; 2, present, asymmetrical (CI = 0.50; RI = 0.86). 10 Two or three rasping spines (rs) occurring below junction of ribbed and smooth region of apical region of the proboscis. 0, absent; 1, present (CI = 1.00; RI = 1.00). 11 Shape of dorsal ligulae. 0 flattened and triangular; 1, conical; 2, forked and triangular; 3, spike-like; 4 curved and triangular; 5, rectangular (CI = 0.80; RI = 96). 84

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12 Tearing hooks (th) occurring in the apical region of the proboscis. 0, absent; 1, present (CI = 1.00; RI = 1.00). 13 Tearing hooks with basiconic sensilla. ?, does not apply; 0, absent; 1, present (CI = 1.00; RI = 1.00). 14 Male antennae. 0, filiform; 1, pectinate; 2, bipectinate (CI = 0.25; RI = 0.68). 15 Female antennae. 0, filiform; 1, pectinate, 2, bipectinate (CI = 0.50; RI = 0.00). 16 Length of labial palp third segment. 0, short (less than half the length of segment 2); 1, medium (half the length of segment 2); 2, long (as long or longer than segment 2) (CI = 0.25; RI = 0.82). 17 Shape of labial palp second segment. 0, ovate; 1, crescent-shape; 2, cylindrical; 3, bent; 4, balloon-shape; 5, boat-shape, wider towards anterior (Fig. 3-3; CI = 0.50; RI = 0.85). 18 Shape of labial palp third segment. 0, rounded; 1, long, finger-like; 2, thumb-like; 3, marble-shaped (Fig. 3-4; CI = 0.44; RI = 0.83). Thoracic characters 19 Hook of the tornus of forewing. 0, absent; 1, present (CI = 0.15; RI = 0.63). 20 Lobe of forewing. 0, absent; 1, present (CI = 0.20; RI = 0.64). Characters of the male genitalia 21 Saccular process. 0, absent; 1, entire; 2, branched or split into two processes (CI = 0.40; RI = 0.65). 22 Shape of saccular process (entire). ?, does not apply; 0, finger-like, pointed, fused to valve; 1, conical, apically truncated; 2, hook-like, thin; 3, triangular prominence; 4, Tshape; 5, thumb-like, triangular 6, thumb-like, setose; 7, small flap; 8, finger-like, pointed, free from valve; 9, asymmetrical, thin, finger-like and thumb-like without setae; A heart-shape; B cylinder with apical node (Fig. 3-5; CI = 0.85; RI = 0.87). 23 Shape of saccular process (branched). ?, does not apply; 0, one branch U-shape and one branch heart-shape; 1, two small points; 2, two thumb-like projecti ons (Fig. 3-6; CI = 1.00; RI = 0.00). 24 Shape of valve. 0, apically rectangular with triangular prominence, anterior lateral edge heart-shape; 1, rectangular; 2, tear-drop shape; 3, triangular; 4 wavy; 5, forked; 6, rounded ventrally, expanding into triangular shape towards dorsum; 7, W-shape; 8, rounded at sides with protruding point; 9, crescent-shape; A M-shape; B trapezoid-shape (Fig. 3-7; CI = 0.65; RI = 0.76). 25 Process of cucullus. 0, absent; 1, present (CI = 0.20; RI = 0.20). 85

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26 Shape of process of cucullus. ?, does not apply; 0, sharp pointed; 1, asymmetrical, double point; 2, symmetrical, double point; 3, M-shape (CI = 1.00; RI = 0.00). 27 Shape of saccus. 0, concave in center; 1, thin and rounded; 2, U-shape with small flaps; 3, V-shape; 4, U-shape without flaps; 5 V-shape, thin; 6, thick and rounded; 7, W-shape; 8, vase-shape; 9, V-shape with two small ventral prominences; A V-shape, split; B compressed triangle (Fig. 3-8; CI = 0.55; RI = 0.55). 28 Manica. 0, membraneous; 1, with fultra; 2, with arellus; with fultra and arellus; 3, setose, with fultra and arellus (CI = 0.50; RI = 0.00). 29 Shape of uncus. 0, hook shape; 1, swollen hook shape; 2, nose-like; 3, long and nose-like (CI = 0.60; RI = 0.78). 30 Hook on uncus apex. 0, absent; 1, present (CI = 0.00; RI = 0.00). 31 Scaphium. 0 membranous; 1, sclerotized (CI = 0.50; RI = 0.50). 32 Shape of scaphium. 0, spoon shape; 1 sclerotized membrane; 2, cross shape; 3, U-shape; 4, wavy parallel lines; 5 crescent shape; 6, sclerotized tube; 7, Y-shape; 8, tongue-shape (CI = 0.67; RI = 0.79). 33 Subscaphium. 0, absent; 1, present, undefined patc hes of sclerotiation; 2, pieces of sclerotization under anal tube; 3, triangular regions of scle rotization (CI = 0.75; RI = 0.83). 34 Coremata. 0, absent; 1, present (CI = 0.25; RI = 0.45). 35 Pattern of sclerotization of sternite VIII. 0, square; 1, no definite shape; 2, divided into two square plates; 3, U-shape; 4, bowtie-shape; 5, inverted Y-shape; 6, V-shape; 7, rectangular with visible antecosta; 8, shield-shape; 9, solid U-shape (CI = 0.90; RI = 0.94). 36 Pattern of sclerotization of tergite VIII. 0, rectangular; 1, H-shape; 2, V-shape; 3, Yshape; 4, triangle shape; 5, square; 6 star-shape (CI = 0.55; RI = 0.83). 37 Shape of dorsal tegumen. 0, rounded, interrupted by circ ular node in center; 1, entire, ring-like; 2, divided with flap in center; 3, thin, M-shape; 4, entire with lateral prominences; 5, rounded, interrupted, without flap in center; 6, wavy (Fig. 3-9; CI = 0.67; RI = 0.91). 38 Shape of uncus base. 0, rounded at sides; 1, thin, square; 2, nose-like; 3, heart-shape; 4, horseshoe shape; 5, triangular; 6, rounded with lateral prominences; 7, W-shape with swollen sides; 8, flattened triangle; 9, flattened square (Fig. 310; CI = 0.56; RI = 0.75). 39 Conjunctiva of uncus and dorsal tegumen. 0, membraneous; 1, contiguous; 2, H-shape; 3, bone-shape; 4, broad, surrounding uncus base; 5, square; 6, large rounded plate urrounding uncus base (CI = 0.45; RI = 0.80). 86

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40 Orientation of phallobase. 0, straight; 1, inflected ventrally at caecum; 2, inflected ventrally at midpoint; 3, inflected dorsally at midpoint; 4, inflected dorsally at caecum (CI = 0.25; RI = 0.48). 41 Shape of caecum. 0, rounded; 1, cylindrical; 2, square; 3, pointed (CI = 0.67; RI = 0.00). 42 Spines of carina. 0, absent; 1, present, extending co mpletely around phallobase; 2, small patch, not extending around phallobase (CI = 0.20; RI = 0.50). 43 Vesica. 0, smooth; 1, rugose, with spiculi; 2, with cone-shaped cornuti (CI = 0.14; RI = 0.50). 44 Abdominal segments 5/6 with specialized scales. 0, absent; 1 present (CI = 0.50; RI = 0.88). Characters of the female genitalia 45 Shape of the antevaginal plate (segment VII). 0, slightly rounded; 1, heart-shape; 2, Yshape; 3, M-shape; 4, divided into two triangul ar plates (Fi. 3-11; CI = 0.57; RI = 0.67). 46 Shape of posterior edge of antevaginal plate. 0, divided into two rectangular plates with ventral edge rounded; 1, divided into two rectangular plates; 2, entire, rectangular; 3, divided into two inverted L-shapes; 4 divided into two triangular plates; 5, divided into two curved plates; 6, trapezoid shape; 7, square with wavy lateral edge; 8, divided into two pentagon shapes; 9, H-shape; A U-shape; B bowtie shape; C divided into two Lshape plates; D divided into two rectangular plates w ith anterior corners sharply pointed; E contiguous, W-shape; F V-shape (Fig. 3-12; CI = 0.74; RI = 0.84). 47 Shape of ductus bursa. 0 cylindrical; 1, wrinkled, sac-like; 2, V-shape; 3, inverted triangle; 4, S-shape (CI = 0.27; RI = 0.35). 48 Shape of postvaginal plate. ?, does not apply; 0, oval-shape; 1, vase-shape; 2, square; 3, heart-shape; 4, M-shape; 5, T shape; 6, flattened oval shape with rounded prominence in center; 7, V-shape; 8, shield-shape; 9, triangular; A small circle; B pentagon shape with stipled base; C mushroom-shape (CI = 1.00; RI = 1.00). 49 Cervical sclerites of the corpus bursa. 0, absent; 1 present (CI = 0.08; RI = 0.54). 50 Shape of cervical scleri tes of the corpus bursa. ?, does not apply; 0, wavy lines; 1, sclerotized patches without definite shape; 2, large rounded area of sclerotization with wavy lines; 3, wavy large sclerotized area; 4, half of posterior sclerotized; 5, oval-shape; 6, sclerotized all around posterior sac of corpus; 7 sclerotized all around anterior sac of corpus; 8, pear-shape; 9, triangular (Fig. 3-13; CI = 0.69; RI = 0.64). 51 Shape of corpus bursa. 0, round, balloon shape; 1, banana shape; 2, peanut shape; 3, thin, teardrop shape; 4, rectangular; 5, heart-shape; 6, clover shape; 7, swollen posterior, saclike anterior; 8, S-shape; 9, long, thin (CI = 0.75; RI = 0.67). 87

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52 Membrane of the corpus bursa. 0, smooth; 1, wrinkled; 2, completely sclerotized; 3, divided into wrinkled posterio r portion and smooth anterior po rtion (Fig. 3-14; CI = 0.19; RI = 0.57). 53 Divison of the antrum and ductus. 0, absent; 1, present (CI = 0.14; RI = 0.60). 54 Shape of antrum. ?, does not apply; 0 cylindrical, completely sclerotized; 1, cylindrical, partially sclerotized; 2, square; 3, vase-shape; 4, triangular; 5 V-shape (CI = 0.83; RI = 0.83). 55 Number of signa. 0, absent; 1, one (CI = 0.67; RI = 0.50). 56 Shape of signa. ?, does not apply; 0, triangular, 1, circle (CI = 1.00; RI = 0.00). 57 Appendix bursa. 0, absent; 1, present (CI = 0.17; RI = 0.64). 58 Appendix bursa. ?, does not apply; 0 membranous; 1, sclerotized (CI = 1.00; RI = 1.00). 59 Shape of appendix bursa. ?, does not apply; 0, cylinder; 1, egg-shape with lateral prominences; 2, flap-like; 3, U shape; 4, ball-shape; 5, kidney-shape; 6, cone-shape; 7, swollen crescent-shape; 8, triangular, nose-like; 9, tube-like (CI = 0.73; RI = 0.40). 60 Ductus seminalis terminati ng at plug of appendix bursa. ?, does not apply; 0, present, small cylinder; 1, triangular (Fig. 3-15; CI = 0.40; RI = 0.40). 61 Plate of intersegmental membrane (IS 9-10). 0, absent; 1, present (CI = 1.00; RI = 1.00). 62 Shape of IS plate. ?, does not apply; 0, triangle; 1 small round node; 2, inverted Y shape; 3, square (CI = 1.00; RI = 0.00). 63 Ornamentation of the ostium bursa. 0, absent; 1, present (CI = 0.13; RI = 0.61). 64 Band of signum. 0, absent; 1, present (CI = 0.50; RI = 0.50). 65 Number of signum bands. ?, does not apply; 0, one; 1, two; 2 three (CI = 1.00; RI = 0.00). 66 Shape of signum band(s). ?, does not apply; 0, ruffled; 1, ring of small lines; 2, wavy lines (CI = 1.00; RI = 0.00). Phylogenetic Analysis of Morphological Data The cladistic analysis resulted in three most parsimonious trees (MPTs) with tree length of 448 steps, a consistency index (CI) of 0.51 and retention index (RI) of 0.74. A strict consensus tree of the three MPTs collapsed nine internal nodes (Fig. 3-16; L = 524, CI = 0.46, RI = 0.71). Sixty of sixty-six char acters were parsimony informativ e and 21% of the data matrix 88

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consisted of missing data. Constant or invari ant characters were in cluded to document state descriptions and distributions for future analyses. The ingroup re lationships were well resolved, except for species relationships within Gonodonta. Despite a poorly resolved Gonodonta clade, the ingroup genera divided into five assembledges: Graphigona + Eudocima Plusiodonta Gonodonta, Oraesia and Calyptra (Fig. 3-16). The monophyly of Calpini was supported by five synapomorphies, three of which were unreversed (CI = 1.00, RI = 1.00) and the jackknife value for the clade was 41 (Table 3-4). This jackknife value was similar to the average jackknife values for other major clades in this an alysis (41 vs. 45). All ingroup genera ( Calyptra Eudocima Graphigona, Gonodonta, Oraesia and Plusiodonta ) were monophyletic and were supported by no fewer than three synapomorphies. The sister relationship of Graphigona to Eudocima was supported by two synapomo rphies and a jackknife value of 45. Diagnostic features for the ingroup genera and other major clades are listed in Table 3-4. The following genera previously associated with Calpini lack ed the unreversed synapom orphies present in all other ingroup genera: Hypsoropha, Phyprosopus and Hemiceratoides The results from this analysis support previous conclu sions that these genera are no t closely related to the other ingroup genera. Given the observe d differences in habitus and proboscis structures in these genera, their current placement within the subfamily is also questionable. Molecular Data and Combined Analyses A parsimony analysis of combined morphologica l and molecular data resulted in six most parsimonious trees with a length of 4572 steps (CI = .28, RI = .27). The strict consensus of six trees was 4643 steps (Fig. 3-17; CI = .28, RI = .25). The data matrix included 638 parsimony informative characters and 47% of the data matrix consisted of missing da ta. The evolutionary model selected for molecular data partitions based on the MUSCLE (Edgar 2004) alignment was the GTR+G. This model was used in conjunc tion with the likelihood -based Mk model for 89

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morphology (Lewis 2001, Ronquist et al. 2005) in the Bayesian an alysis. The first 28,0000 trees of 10, 500, 000 were discarded as burn-in as indicated by graphing the generations in Microsoft Office Excel 2003. The majority rule topology was recovered from the Bayesian analysis (Fig. 3-19) in PAUP* 4.0 (Swofford 2000). The three result ing topologies were compared for overall similarity using the algorithm and software program ( implemented as a Java applet at http://www.mrc-bsu.cam .ac.uk/personal/thomas/phylo-comparison/ comparison_page.html. ) described in Nye et al. (2006). Because this approach assumes topologies under comparison have equal branch nu mbers, taxa were trimmed from the strict consensus tree based on morphological data to ma tch the taxon sampling in the morphological + molecular dataset. The strict consensus t oplogy resulting from the parsimony analysis of molecular and morphological data was 59% similar to the stri ct consensus topology based on morphological data. Although both analyses recovered a monophyletic ingroup, differences between the topologies were observe d (compare Figs. 3-16 and 3-18). Plusiodonta and Eudocima remained monophyletic in the parsimony analysis of combined data; other relationships between ingroup genera were in congruent with respect to the alternative phylogenetic hypothesis based on morphological data alone. The topology based on combined data and parsimony analysis placed Gonodonta and some Oraesia species as sister genera and Calyptra basal to Eudocima (Fig. 3-18). In the parsimony an alysis based on morphological data, the arrangement is different: Calyptra and Oraesia are sister genera and the Graphigona + Eudocima clade is sister to the rema ining ingroup genera (Fig. 3-16) In the parsimony analyses of separate and combined data, the composition of the ingroup wa s stable to removal of outgroup taxa and the outgroup relationships are largel y similar in both resulting topologies. The parsimony analyses were both 42% similar to the topology resulting from the Bayesian analysis 90

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of combined data. The Bayesian analysis do es not support the monophyly of Calpini and several species (e.g., Hypsoropha spp. and Phyprosopus callitrichoides ) considered to be outgroup taxa were placed within the ingroup clade (Fig. 3-19). Additionally, support for monophyly of all ingroup genera is lost in the Ba yesian topology and taxa are erratically placed in clades with genera thought previously to be distantly related (e.g., Eudocima tyrannus). Branch support values were generally low in both combined analyses, with few nodes supported by values greater than 80 for all three support measures (Figs. 3-18 an d 3-19). Due to large amounts of missing data and a reduction in taxon sampling [w hen compared to the morphological matrix], the combined analyses in this study should be considered preliminary. The topology resulting from the Bayesain analysis was largely incongru ent with respect to the topologies resulting from parsimony analyses. This suggests that alternative modeling stratageies may need to be explored with the combined data set prior to analyzing th e data in a Bayesian framework in the future. Evolution of Feeding Behaviors and Complementary Analysis Examination of adult feeding records revealed that reports were available for 39 taxa included in the cladistic analysis (60% of all term inal taxa). These feeding reports and fruit hosts are summarized in Table 3-4. The binary feed ing behaviors were optimized onto the strict consensus phylogeny resulting from the parsimony analysis based on morphological data. This approach was taken due to the reduction of taxa and large amounts of missing data in the combined analyses. All taxa included in this analysis of adult feedi ng behavior have been reported taking nectar, fruit juice, or feeding at fruit juice baits (Fig. 3-17). Many of the taxa included in this analysis are also primary pi ercers of thick-skinned fruits. Outgroup taxa, Anomis spp. and Scoliopteryx libatrix are primary piercers of thick-ski nned fruits and fi ve out of six ingroup genera have documented pr imary piercing reports for at le ast two species; adult feeding behaviors for Graphigona have not been published. Primary pi ercing of thick-skinned fruits has 91

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been reported for eight Eudocima species included in this analysis (Table 3-4). All other primary piercers of thick-skinned fruits are considered secondary piercers of hard-skinned fruits until consistent observational data demonstrating otherw ise emerges. According to this analysis, one independent incident of tear f eeding has occurred in the subfamily Calpinae. Tear feeding on sleeping birds was reported for Hemiceratoides hieroglyphica in Madagascar by Hilgartner et al. (2007). The phylogenetic analysis included si xteen of the seventeen described Calyptra species (Bnziger 1983). Of the sixteen incl uded in this analysis, males of ten Calyptra species have been reported feeding on blood under experimental or natural conditions. A single report of Calyptra lata feeding on blood was observed in July (Zaspel, unpublished data 2008). This record for C. lata has been included in this analysis but it has been noted that it was a single occurrence (Fig. 3-17). Equivocal cycling in MacClade 4.0 (Maddison and Maddison 2000) was used to determine that skin piercing and blood f eeding is the most derive d feeding type within the Calpini and that hematophagy has evolved four times within Calyptra Conclusions The results from this study support the hypothesis that hematophagy in the genus Calyptra evolved from the fruit-piercing habit (B nziger 1971) as opposed to tear feeding (Downes 1973, Hilgartner et al. 200 7). These results also support a directional progression of feeding types from nectar feeding to fruit pi ercing, culminating in skin piercing and blood feeding hypothesized by Bnziger (1971). This work suggests blood feeding has evolved multiple times within the genus Calyptra New blood feeding records have been been described in recent literature (Zaspel et al. 2007) a nd recorded on recent collecting expeditions (e.g., C. lata Zaspel unpublished field obser vation 2008). Thus, it is po ssible that blood feeding does occur in other Calyptra species but has not ye t been observed. Although these results are based 92

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soley on the morphological data matrix, the hypothe sis that hematophagy is derived from fruit piercing in these moths is also supported by th e parsimony analysis of combined morphological and molecular data. The Bayesian analysis pr oduced an alternative phylogenetic hypothesis that does not support a monophyletic Calpini; both combined analyses should be considered preliminary at this time. Results from the analysis based on morphol ogical data suggest Calpini is monophyletic and is supported by five synapomorphies. Three of these are unreversed, shared characters of the proboscis. With the exception of the genus Graphigona, all remaining genera within Calpini are monophyletic and are supported by at least th ree synapomorphies. Whether or not Graphigona and Eudocima are synonymous needs further investiga tion. Tear feeding, or feeding on wounds and other secretions has also evolved multiple times within Lepidoptera (Fig. 3-2: A); however, fruit piercing species within the Calpinae have not been observed feeding on tears. If the observed tear-feeder Hemiceratoides hieroglyphica is in fact a member of Calpinae, it will represent an independent origin of te ar feeding within the the subfamily. The lack of variation in pr oboscis characters among ingr oup genera is surprising and suggests proboscis morphology is not completely correlated with feeding type. All species within Gonodonta, Oraesia and Calyptra respectively, have identical proboscis armature, yet blood-feeding species have only been documented in the genus Calyptra Thus, there are no specialized structures present in the ten docum ented blood-feeders. Additionally, species in genera from other tribes within the subfamily (e.g., Anomis and Scoliopteryx ) used as outgroups in this study are documented primary piercers, yet they lack the tearing h ooks, erectile barbs, and rasping spines found in all ingroup taxa. Othe r distantly related noctu id genera presently considered catocalines also pierce fruit yet have far weaker and less armoured proboscides. It 93

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94 is likely that fruit piercing in adult moths has evolved multiple times within the family Noctuidae as a result of ecological convergence.

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Table 3-1. Specimens examined B. Taxon Classification Sampling locality Molecular partitions Voucher specimen # (sex, slide number, collection) COI 28S Anomis flava (Walker) Anomini Austra lia (F-JMZ462, HFay) COI JMZD006 A. mesogona (Walker) Anomini Taiwan (M-JMZ431, FLMNH) COI JMZE002 A. mesogona Anomini Taiwan (F-JMZ432, FLMNH) Calyptra albivirgata (Hampson) Calpini China (M-JMZ359, NMNH) COI JMZA006 Calyptra albivirgata Calpini Japan (F-JMZ503, NHM) C. bicolor (Moore) Calpini Nepal (M-JMZ348, HB-1755) COI JMZA004 C. bicolor Calpini Nepal (F-JMZ349, HB) C. canadensis (Bethune) Calpini U.S.A. (M-JMZ374, AMNH) 28S JMZ020-2 C. canadensis Calpini U.S.A. (F-JMZ374, AMNH) C. eustrigata (Hampson) Calpini Ma laysia (M-JMZ331, HB) C. eustrigata Calpini Thailand (F-JMZ332, HB) C. fasciata (Moore) Calpini Nepal (M-JMZ329, MF) C. fasciata Calpini Nepal (F-JMZ330, MF) C. fletcheri (Berio) Calpini Nepal (M-JMZ352, HB-1878) C. gruesa (Draudt) Calpini China (M-JMZ357, NMNH) C. gruesa Calpini Japan (F-JMZ505, NHM) C. hokkaida Wileman Calpini Japan (F-JMZ492, UMD) C. hokkaida Calpini China (Bnziger, 1983; Figs. 11, 76-77) C. lata (Butler) Calpini Korea (F-JMZ358, NMNH) COI 28S JMZA003, JMZ018-1 C. lata Calpini Russia (M-JMZ495, FLMNH) C. minuticornis (Guene) Calpini Nepal (M-JMZ351, MF) C. minuticornis Calpini Thailand (F-JMZ354, HB) C. ophideroides (Guene) Calpini Himalaya [sic.] (F-JMZ335, NMNH) C. ophideroides Calpini India (Bnziger, 1983; Figs. 15, 78-83) 95

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Table 3-1. Continued Taxon Classification Sampling locality Molecular partitions Voucher specimen # (sex, slide number, collection) COI 28S C. orthograpta (Butler) Calpini China (M-JMZ347, NMNH) C. orthograpta Calpini Thailand (F-JMZ346, HB) C. parva Bnziger Calpini Thailand (M-JMZ344, HB-2784) C. parva Calpini Thailand (F-JMZ345, HB) C. pseudobicolor Bnziger Calpini Nepal (F-JMZ341, HB-1779) C. pseudobicolor Calpini Nepal (M-JMZ342, HB-1803) C. subnubila (Prout) Calpini Indonesia (F-JMZ350) C. subnubila Calpini Indonesia (M-Bnziger, 1983; Figs. 17, 36-37) C. thalictri (Borkhausen) Calpini U nknown (M-JMZ334, FLMNH) COI 28S JMZA002, JMZ017-1 C. thalictri Calpini Russia (F-JMZ360, NMNH) Eudocima aurantia (Moore) Calpini Australia (M-JMZ417, MF) COI JMZD012 E. aurantia Calpini Australia (F-JMZ418, MF) E. anguina (Schaus) Calpini Peru (F-JMZ500, NHM) E. boseae (Saal.) Calpini Madagascar (F-JMZ498, NHM) E. boseae Calpini Madagascar (M-JMZ499, NHM) E. cocalus (Cramer) Calpini Queensland (M-JMZ419, HFay) E. cocalus Calpini Queensland (F-JMZ420, HFay) E. dividens (Walker) Calpini Unknown (M-JMZ467, NMNH) E. dividens Calpini Luzon (F-JMZ468, NMNH) E. homaena (Hbner) Calpini Taiwan (F-JMZ429, FLMNH) E. homaena Calpini Taiwan (M-JMZ430, FLMNH) E. jordani (Holland) Calpini Queensland (F-JMZ425, HFay) COI JMZD009 E. jordani Calpini Queensland (M-JMZ426, HFay) E. materna (L.) Calpini Malawi (F-JMZ400, NMNH) COI JMZ2091 E. materna Calpini Malawi (M-JMZ401, NMNH) 96

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Table 3-1. Continued Taxon Classification Sampling locality Molecular partitions Voucher specimen # (sex, slide number, collection) COI 28S E. phalonia (L.) Calpini Queensland (M-JMZ463, HFay) E. phalonia Calpini Queensland (F-JMZ464, HFay) E. procus (Cramer) Calpini Peru (F-JMZ514, HMNH) E. salaminia (Cramer) Calpini Indonesia (M-JMZ395, NMNH) COI JMZA008 E. salamin Calpini New Guinea (F-JMZ396, NMNH) E. tyrannus (Guene) Calpini China (M-JMZ465, NMNH) 28S JMZ023-3 E. tyrannus Calpini Nepal (F-JMZ466, MF) Gonodonta correcta Walker Calpini Brazil (F-JMZ516, NMNH) COI MHAUA414-60703 G. correcta Calpini Brazil (M-JMZ517, NMNH) G. incurva (Sepp) Calpini U.S.A. (M-JMZ470, FLMNH) COI MHAUA078-57660 G. incurva Calpini Peru (F-JMZ509, NHM) G. indentata (Hampson) Calpini Venezuela (F-JMZ382, NMNH) COI MHAUA005 -17237 G. indentata Calpini Brazil (M-JMZ383, NMNH) G. mexicana Schaus Calpini Bolivia (F-JMZ402, NMNH) G. mexicana Calpini Ecuador (M-JMZ403, NMNH) G. nutrix (Cramer) Calpini U.S.A. (M-JMZ445, FLMNH) 28S JMZ046-3 G. nutrix Calpini U.S.A. (F-JMZ446, FLMNH) G. parens (Guene) Calpini Panama (F-JMZ455, NMNH) COI JMZD002 G. parens Calpini Panama (M-JMZ456, NMNH) G. sicheas (Cramer) Calpini Brazil (M-JMZ451, FLMNH) COI MHAUA015-27415 G. sicheas Calpini D.R. (F-JMZ452, FLMNH) G. sinaldus (Guene) Calpini U.S.A. (M-JMZ449, FLMNH) COI BLPBD566 G. sinaldus Calpini U.S.A. (F-JMZ450, FLMNH) G. unica Neumoegen Calpini U.S.A. (F-JMZ447, FLMNH) G. unica Calpini U.S.A. (M-JMZ448, FLMNH) 97

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Table 3-1. Continued Taxon Classification Sampling locality Molecular partitions Voucher specimen # (sex, slide number, collection) COI 28S G. uxor (Cramer) Calpini Costa Ri ca (M-JMZ489, UMD) COI 28S MHAU415, JMZ007 G. uxor Calpini Venezuela (F-JMZ405, NMNH) Graphigona regina (Guene) Calpini Costa Rica (F-JMZ510, NHM) Graphigona regina Calpini Guatemala (M-JMZ511, NHM) Hemiceratoides hieroglyphica (Saal.) I.S. Malawi (M-JMZ61, NMNH) Hemiceratoides hieroglyphica I.S. Liberia (F-JMZ62, NMNH) Hemiceratoides sittaca Karsch I.S. Uganda (M-JMZ518, NMNH) COI JMZC002 Hypsoropha hormos Hbner I.S. U.S.A. (F-JMZ476, FLMNH) 28S JMZ006-1 Hypsoropha hormos I.S. U.S.A. (M-JMZ491, FLMNH) Hypsoropha monilis Fabricius I.S. U.S.A. (M-JMZ474, FLMNH) 28S JMZ002-1 Hypsoropha monilis I.S. U.S.A. (F-JMZ475, FLMNH) Oraesia argyrosigna Moore Calpini Tanzania (M-JMZ391, NMNH) O. argyrosigna Calpini Nepal (F-JMZ394, MF) O. emarginata (Fabricius) Calpini Malaysia (F-JMZ388, NMNH) COI JMZB11 O. emarginata Calpini Sri Lanka (M-JMZ389, NMNH) O. excavata (Butler) Calpini Japan (M-JMZ365, NMH) O. excavata Calpini Japan (F-JMZ366, NMH) O. excitans (Walker) Calpini Mexico (F-JMZ379, NMNH) COI 28S MHAB508, JMZ008 O. excitans Capini Costa Rica (M-JMZ486, UMD) O. glaucohelia (Hampson) Calpini Bolivia (M-JMZ378, NMNH) O. glaucohelia Calpini Brazil (F-JMZ507, NHM) O. nobilis Felder and Rogenhofer Calpini Brazil (F-JMZ363, NMNH) COI 28S MHAB677, JMZ009 O. nobilis Calpini Costa Rica (M-JMZ487, UMD) O. provocans Walker Calpini Malawi (F-JMZ369, NMNH) O. rectistria Guene Calpini India (F-JMZ370, NMNH) COI 28S JMZF11, JMZ011-1 O. rectistria Calpini Nepal (M-JMZ485, FLMNH) 98

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Table 3-1. Continued Taxon Classification Sampling locality Molecular partitions Voucher specimen # (sex, slide number, collection) COI 28S O. serpans Schaus Calpini Peru (M-JMZ508, NHM) COI 28S MHAB514, JMZ010 O. striolata Schaus Calpini Bolivia (F-JMZ376, NHM) O. striolata Calpini Peru (M-JMZ377, NHM) O. triobliqua (Saalmller) Calpini Zimbabwe (M-JMZ337, NMNH) O. triobliqua Calpini Madagascar (F-JMZ506, NHM) Phyprosopus callitrichoides Grote I.S. U.S.A. (F-JMZ479, FLMNH) 28S JMZ003-1 Phyprosopus callitrichoides I.S. U.S.A. (M-JMZ484, FLMNH) Plusiodonta casta (Butler) Calpini Japan (M-JMZ511, NHM) 28S JMZ045-3 P. casta Calpini Japan (F-JMZ512, NHM) P. coelonota (Kollar) Calpini Taiwan (M-JMZ384, NMNH) COI JMZC008 P. coelonota Calpini Taiwan (F-JMZ385, NMNH) P. compressipalpus Guene Calpini U.S.A. (F-JMZ458, FLMNH) COI 28S JMZF12, JMZ012-1 P. compressipalpus Calpini U.S.A. (M-JMZ483, FLMNH) P. dimorpha Robinson Calpini Costa Rica (F-JMZ408, NMNH) COI JMZE004 P. dimorpha Calpini Fiji (M-JMZ409, NMNH) P. incitans (Walker) Calpini Mexico (F-JMZ386, NMNH) P. incitans Calpini Argentina (M-JMZ387, NMNH) P. miranda Schaus Calpini Argentina (F-JMZ406, NMNH) P. miranda Calpini Costa Rica (M-JMZ407, NMNH) P. repellens (Walker) Calpini Costa Rica (M-JMZ415, NMNH) P. repellens Calpini Mexico (F-JMZ416, NMNH) Scoliopteryx libatrix (L.) Scoliopterygini De nmark (M-JMZ381, FLMNH) COI 28S JMZA001, JMZ001-1 Scoliopteryx libatrix Scoliopterygi ni Denmark (F-JMZ404, FLMNH) 99

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100 Table 3-2. PCR conditions and sequences of primers used. ________________________________________________________________________ COI: 94 C/1 min; 5 cycles of: 94 C/30 s, 45 C/40 s, 72.0 C/30 s; 30 cycles of: 94 C/30 s; 51 C/40 s, 72.0 C/1 min; final extension of: 72.0 C/10 min. 28S: 94 C/2 min 30 sec; 94 C/30 s; 65 C/30 s; 72.0 C/30 s; 2 x 40; 72.0 C/2 min. ________________________________________________________________________ Primers Sequence from 5 to 3 ________________________________________________________________________ COI DNA Barcode Segment: LepF1 5' ATTCAACCAATCATAAAGATATTGG -3' LepR1 5' TAAAC TTCTGGATGTCCAAAAAATCA -3' MLepF1 5' GCTTTCCCACGAATAAATAATA -3' MLepR1 5' CCTGTTCCAGCTCCATTTTC -3' 28S D2 Loop: 28S_D2F1 5' GAG TAC GTG AAA CCG TTC AG 3' 28S_D2R1 5' CTG ACC AGG CAT AGT TCA C 3' ________________________________________________________________________

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Table 3-3. Known feeding behavior reports for Calpini and related genera included in complementary analyses (U = unknown, NF = nectar/fruit sucking, PTF = primary piercer of thick-skinne d fruits, SP = secondary piercer of hard-skinned fruts, PHF = primary piercer of hard-skinned fruits, BF = skin piercer and blood feeder, TF = tear feeder). Genus species Feeding Behavior(s) Host(s) Reference(s) Anomis flava PTF Grape, raspberry Yoon & Lee 1974, Bnziger 1982, 1987 mesogona PTF Grape, peach, plum Nomura & Hattori 1967, Yoon & Lee 1974, Bnziger 1982, 1987 Calyptra bicolor PTF Mandarin, raspberry Bnziger 2007 bicolor BF Human Bnziger 1989 eustrigata PTF Apple, mandarin Bnziger 2007 eustrigata BF Water buffalo, tapir Bnziger 1968, 1975 fasciata PTF Apple, mandarin Bnziger 2007 fasciata BF Human elephant Bnziger 1986, 1989 fletcheri PTF Apple, mandarin Bnziger 2007 fletcheri BF Human Bnziger 1989 101 gruesa PTF Peach, grape, apple Hattori 1962 hokkaida PTF Peach, grape Hattori 1962 lata PTF Peach, grape, apple Hattori 1962, Yoon & Lee 1974 lata BF Human Pers. observation 2008 minuticornis PTF Apple, mandarin Bnziger 2007 minuticornis BF Human elephant Bnziger 1986 ophideroides PTF Apple, mandarin Hargreaves 1934, Bnziger 2007 ophideroides BF Human Bnziger 1989 orthograpta PTF Apple, mandarin Bnziger 2007 orthograpta BF Human elephant Bnziger 1986, 1989

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Table 3-3. Continued Genus species Feeding Behavior(s) Host(s) Reference(s) Calyptra parva PTF Apple, mandarin Bnziger 2007 parva BF Human Bnziger 1989 pseudobicolor PTF Apple, mandarin Bnziger 2007 pseudobicolor BF Human Bnziger 1989 thalictri PTF, Grape, citrus Hargreaves 1934, Yoon & Lee 1974 thalictri BF Human Zaspel et al. 2007 Eudocima aurantia PHF Longan, mandarin Bnziger 1982, Fay & Halfpapp 1999 cocalus PHF Longan, mandarin Fay & Halfpapp 1999 homanea PHF Longan, mandarin Fay & Halfpapp 1999 jordani PHF Longan, mandarin Fay & Halfpapp 1999 materna PHF Longan, mandarin Fay & Halfpapp 1999 phalonia PHF Longan, mandarin Fay & Halfpapp 1999 102 salaminia PHF Longan, mandarin Fay & Halfpapp 1999 tyrannus PHF Longan, mandarin Fay & Halfpapp 1999 Gonodonta incurva PTF Citrus Todd 1959 nutrix PTF Citrus Todd 1959 uxor PTF Citrus Bruner et al. 1945

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Table 3-3. Continued Genus species Feeding Behavior(s) Host(s) Reference(s) Hemiceratoides hieroglyphica TF Newtonia, Magpie Robin Hilgartner et al. 2007 Hypsoropha hormos NF Fruit bait Pers. observation monilis NF Fruit bait Pers. observation Oraesia argyrosigna PTF Mandarin Bnziger 1982 emarginata PTF Grape Yoon & Lee 1974 excavata PTF Grape Yoon & Lee 1974 provocans PTF Citrus Hargreaves 1934 rectristria PTF Plum Bnziger 1987, pers. observation triobliqua PTF Citrus Hargreaves 1934 Phyprosopus callitrichoides NF Fruit bait Personal observation Plusiodonta 103 casta PTF Peach, grapes Maff 1990 coelonota PTF Peach, grapes, guava Bnziger 1982, Maff 1990 Scoliopteryx libatrix Raspberry, grape Yoon & Lee 1974, pers. observation

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Table 3-4. Character support for major clades. Indicates ge nera placed in tribe Calpini (sensu Zaspel & Branham 2008). Clade Character: stat e: CI, RI Jacknife Value A Anomis + ( Scoliopteryx + ( Hyproropha + Phyprosopus ) + (Hemiceratoides ) + [1: 1: 1.00, 1.00] 100 ( Graphigona + ( Eudocima + ( Plusiodonta + ( Gonodonta + ( Calyptra + Oraesia )))))) [2: 2: 1.00, 1.00] [25: 0: 0.20, 0.20] B Scoliopteryx + ( Hyproropha + Phyprosopus ) + ( Hemiceratoides ) + [1: 1: 1.00, 1.00] 18 ( Graphigona + ( Eudocima + ( Plusiodonta + ( Gonodonta + ( Calyptra + Oraesia )))))) [27: 3: 0.55, 0.55] [41: 0: 0.67, 0.00] [53: 0: 0.14, 0.60] C Hemiceratoides + ( Graphigona + ( Eudocima + ( Plusiodonta + [4: 1: 1.00, 1.00] 48 ( Gonodonta + ( Calyptra + Oraesia ))))) [20: 1: 0.20, 0.64] D Graphigona + ( Eudocima + ( Plusiodonta + ( Gonodonta + ( Calyptra + Oraesia )))) [7: 1: 0.50, 0.83] 41 [8: 1: 1.00, 1.00] [12: 1: 1.00, 1.00] [26: 0: 1.00, 0.00] [43: 1: 0.14, 0.50] 104 E Plusiodonta + ( Gonodonta + ( Calyptra + Oraesia )) [5: 0: 1.00, 1.00] 29 [21: 1: 0.40, 0.65] F Gonodonta + ( Calyptra + Oraesia ) [16: 0: 0.25, 0.82] 18 G Hypsoropha sp. + ( Hypsoropha sp. + Phyprosopus ) [1: 0: 1.00, 1.00] 65 [2: 1: 1.00, 1.00] [6: 1: 1.00, 1.00] H Graphigona + ( Eudocima ) [4: 3: 1.00, 1.00] [17: 5: 0.50, 0.85] 45 I Calyptra + Oraesia [32: 0: 0.67, 0.79] 34 [39: 2: 0.45, 0.80] J Hemiceratoides [9: 2: 0.50, 0.86] 98

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Table 3-4. Continued Clade Character: stat e: CI, RI Jacknife Value J Hemiceratoides [27: 7: 0.55, 0.55] [31: 0: 0.50, 0.50] [35: 4: 0.90, 0.94] K Eudocima [10: 1: 1.00, 1.00] 15 [11: 3: 0.80, 0.96] [13: 1: 1.00, 1.00] [29: 2: 0.60, 0.78] [39: 5: 0.45, 0.80] L Plusiodonta [3: 1: 1.00, 1.00] 38 [11: 2: 0.80, 0.96] [54: 2: 0.83, 0.83] M Gonodonta [11: 1: 0.80, 0.96] 66 [18: 3: 0.44, 0.83] [33: 2: 0.75, 0.83] [44: 1: 0.50, 0.88] [47: 2: 0.27, 0.35] N Oraesia [9: 1: 0.50, 0.86] 64 [24: 8: 0.65, 0.76] [37: 2: 0.67, 0.91] [46: 8: 0.74, 0.84] [57: 1: 0.17, 0.64] [60: 1: 0.40, 0.40] O Calyptra [22: 5: 0.85, 0.87] 7 [36: 6: 0.55, 0.83] [40: 3: 0.25, 0.48] 105

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Figure 3-1. Blood-feeding moth, Calyptra thalictri 106

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Figure 3-2. Proposed hypotheses fo r the evolution of tear feed ing and blood feeding within Lepidoptera. 107

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Figure 3-3. Shape of labial palp segment II, Character 17: Taxon voucher number (state, condition). A S. libatrix JMZ381 (0, ovate); B A. mesogona JMZ431 (1, crescentshape); C H. monilis JMZ475 (2, cylindrical); D P. callitrichoides JMZ478 (3, bent); E O. emarginata JMZ388 (4, balloon-shape); F E. homanea JMZ430 (5, boat-shape, wider towards anterior). 108

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Figure 3-4. Shape of labial palp segment III, Character 18: Taxon (state, condition). A C. albivirgata JMZ359 (0, rounded); B S. libatrix JMZ381 (1, long, finger-like); C C. eustrigata JMZ331 (2, thumb-like); D G. incurva JMZ470 (3, marble-shaped), E P. callitrichoides JMZ478 (4, cylinder). 109

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Figure 3-5. Shape of saccular process (entire) Character 22: Taxon (state, condition). A P. casta JMZ513 (0, finger-like, point ed, fused to valve); B S. libatrix JMZ381 (1, cone-shape, apically truncated); C G. correcta JMZ517 (2, hook-like, thin); D H. hormos JMZ491 (3, triangular prominence); E C. canadensis JMZ374 (4, T-shape); F C. gruesa JMZ357 (5, thumb-like, triangular); G P. compressipalpus JMZ483 (6, thumb-like, setose); H P. coelonota JMZ384 (7, small flap); I C. thalictri JMZ482 (8, finger-like, pointed, free from valve); J C. parva HB2784 (9, asymmetrical, thin, finger-like and thumb-like without setae); K C. pseudobicolor HB1803 (A, heartshape); L O. argyrosigna JMZ391 (B, cylinder w ith apical node). 110

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Figure 3-6. Shape of saccular process = SaP (branc hed), Sa = saccus, Character 23: Taxon (state, condition). For C. hokkaida (0, one branch U-shape and one branch heart-shape) see Bnziger 1883, Fig.6; A O. rectristria JMZ485 (1, two small points); B P. callitrichoides JMZ478 (2, two thumblike projections). 111

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Figure 3-7. Shape of valve, Character 24: Taxon (state, condition). A P. compressipalpus JMZ483, right valve (0, apically r ectangular with triangular promin ence, anterior lateral edge heart-shape); B O. rectristria JMZ485, right valve (1, rectangular); C P. casta JMZ513, left valve (2, tear-drop shape); D E. boseae JMZ499, left valve (3, triangular); E H. hieroglyphica JMZ361, left valve (4, wavy); F S. libatrix JMZ381, left valve (5, forked); G C. orthograpta JMZ346, left valve (6, rounded ventrally, expandi ng into triangular shape towards dorsum); H G. sinaldus JMZ449, left valve (7, W-shape); IO. argyrosigna JMZ391, right valve (8, rounded at sides with protruding point); J P. callitrichoides JMZ478, left valve (9, crescent-shape); K E. tyrannus JMZ465, left valve (A, M-shape); L A. mesogona JMZ431, left valve (B, trapezoidshape). 112

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Figure 3-8. Shape of saccus, Character 27: Taxon (state, condition). A S. libatrix JMZ381 (0, concave in center); B O. serpans JMZ508 (1, thin and rounded); C G. correcta JMZ517 (2, U-shape with small flaps); D G. regina JMZ511 (3, V-shape); E G. sinaldus JMZ449 (4, U-shape without flaps); F P. compressipalpus JMZ457 (5, Vshape, thin); G E. boseae JMZ499 (6, thick and rounded); H H. hieroglyphica JMZ361 (7, w-shape); I O. triobliqua JMZ338 (8, vase-shape); J E. cocalus JMZ419 (9, V-shape with two small ventral prominences); K H. hormos JMZ491 (A, V-shape, split); L H. monilis JMZ474 (B, compressed triangle). 113

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Figure 3-9. Shape of dorsal tegumen, Character 37: Taxon (state, condition). A O. serpans JMZ508 (0, rounded, interrupted by circular node in center); B G. correcta JMZ517 (1, entire, ring-like); C P. casta JMZ513 (2, divided with flap in center); D A. mesogona JMZ431 (3, thin, M-shape); E G. regina JMZ511 (4, entire with lateral prominences); F H. hieroglyphica JMZ361 (5, rounded, interrupted, without flap in center); G P. callitrichoides JMZ478 (6, wavy). 114

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Figure 3-10. Shape of uncus base 38: Taxon (state, condition). A P. casta JMZ513 (0, rounded at sides); B G. correcta JMZ517 (1, thin, square); C E. boseae JMZ499 (2, noselike); D G. indentata JMZ383 (3, heart-shape); E S. libatrix JMZ381 (4, horseshoe shape); F E. cocalus JMZ419 (5, triangular); G P. compressipalpus JMZ457 (6, rounded with lateral prominences); H E. phalonia JMZ463 (7, W-shape with swollen sides); I G. parens JMZ456 (8, flattened triangle); J A. mesogona JMZ431 (9, flattened square). 115

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Figure 3-11. Shape of the posterior edge of the antevaginal plate (segment VII), Character 45: Taxon (state, condition). A H. hieroglyphica JMZ362 (0, slightly rounded); B G. invurva JMZ509 (1, heart-shape); C G. regina JMZ510 (2, Y-shape); D C. lata JMZ495 (3, M-shape); E P. miranda JMZ406 (4, divided into tw o triangular plates). 116

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Figure 3-12. Shape of the posterior edge of se gment VIII, Character 46 : Taxon (state, condition). A C minuticornis JMZ354, ventral view (0, divided in to two rectangular plates with ventral edge rounded); B H. hieroglyphica JMZ362, ventral view (1, divided into two rectangular plates); C G. incurva JMZ509, ventral view (2, entire, rectangular); D P. coelonota JMZ512, ventral view (3, divided in to two inverted L-shapes); E S. libatrix JMZ404, ventral view (4, divided into two triangular plates); F C. albivirgata JMZ503, ventral view (5, divided into two curved plates); G E. phalonia JMZ464, ventral view (6, trapezoid shape); H E. cocalus JMZ420, ventral view (7, square with wavy lateral edge); I H. monilis JMZ475, ventral view (8, Y-shape); J E. boseae JMZ498, ventral view (9, H-shape); K C. canadensis JMZ375, ventral view (A, U-shape); L A. mesogona JMZ432, ventral view (B, bowtie shape); M P. compressipalpus JMZ520, ventral view (C, divided into two L-shape plates); N C. parva JMZ345, ventral view (D, divided into tw o rectangular plates with anterior corners sharply pointed); O E. aurantia JMZ418, ventral view (E, contiguous, Wshape); P E. homanea JMZ429, ventral view (F, V-shape). 117

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Figure 3-13. Shape of cervical sclerites of the corpus bursa, Character 50: Ta xon (state, condition). A P. miranda JMZ406 (0, wavy lines); B P. coelonota JMZ512 (1, sclerotized patches without definite shape); C O. excavata JMZ366 (2, large rounded area of sclerotiza tion with wavy lines); D G. uxor JMZ405 (3, large sclerotized area); E E. tyrannus JMZ466 (4, half of poste rior sclerotized); F E. boseae JMZ498 (5, oval-shape); G C. albivirgata JMZ503 (6, sclerotized all around posterior sac of corpus); H C. orthograpta JMZ347 (7, sclerotized all around anterior sac of corpus); I C. pseudobicolor HB1779 (8, pear-shape); J C. subnubila JMZ350 (9, triangular). 118

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Figure 3-14. Shape of the corpus bursa, Ch aracter 52: Taxon (sta te, condition). A O. excavata JMZ366 (0, round, balloon shape); B E. salaminia JMZ396 (1, banana shape); C P. miranda JMZ406 (2, peanut shape); D P. incitans JMZ386 (3, thin, teardrop shape); E G. indentata JMZ382 (4, rectangular); F G. sinaldus JMZ450 (5, heartshape); G G. sicheas JMZ452 (6, clover shape); H C. orthograpta JMZ347 (7, swollen posterior, saclike anterior); I O. argyrosigna JMZ394 (8, S-shape); J P. repellens JMZ416 (9, long, thin). 119

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Figure 3-15. Shape of appendix bursa (AB), Character 60: Taxon (state, condition). A O. striolata JMZ376 (0, cylinder); B O. emarginata JMZ388 (1, egg-shape with lateral prominences); C O. triobliqua JMZ506 (2, flap-like); D O. rectristria JMZ370 (3, U shape); E O. argyrosigna JMZ394 (4, ball-shape); FO. glaucochelia JMZ507 (5, kidney-shape); G O. provocans JMZ369 (6, cone-shape); H C. eustrigata JMZ343 (7, swollen, crescent-shape); I E. materna JMZ400 (8, triangular, nose-like); J G. correcta JMZ416 (9, tube-like). 120

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Figure 3-16. Strict consensus tree of three most parsimonius rearrangements for 65 taxa based on sixy-six morphological characters (L = 524, CI = 0.46, RI = 0.71). Numbers above branches are jackknife values for major clades; numbers below branches are synapomorphies for major clades. Black vertical line indica tes outgroup taxa of the tribe Calp ini, red vertical line indicates ingroup taxa of tribe Calpini; remaining taxa ar e outgroup taxa representi ng other genera in the subfamily Calpinae. 121

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Figure 3-17. Evolution of adult feeding behavior s in Calpini. Characters are mapped onto the strict consensus phylogeny using presence/ab sence characters: A = nectar feeding, B = secondary fruit piercing, C = primary fr uit piercing (thick-skinned fruits), D = primary fruit piercing (hard-sk inned fruits), E = tear f eeding F = skin piercing and blood feeding. Skin piercing and blood feedi ng as a binary charac ter is optimized to show the multiple origins of hematophagy in the genus Calyptra Indicates single occurrence of blood feeding. 122

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Figure 3-18. Preliminary strict consensus tree of six most parsimonius rearrangements for 34 taxa based on combined data set (66 morphological characters and segments of COI (665 bp) and 28S (696 bp) genes; L = 4643, CI = 0.28, RI = 0.25). Numbers above branches are nonparametric bootstrap values for clades; numbers below branches are jackknife calculations for clades. Solid line indicates ingroup taxa of the tribe Calpini; remaining taxa are outgroup taxa re presenting other genera in the subfamily Calpinae. 123

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Figure 3-19. Preliminary Bayesian analysis resulting from a simultaneous analysis of all data partitions for 34 taxa. Majority rule c onsensus of topology generated via MrBayes with 10, 500, 000 generations using model GTR + G for molecular data sets and the Mk Model for morphology. Numbers above branches are posterior probabilities. 124

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CHAPTER 4 WORLD CHECKLIST OF TRIBE CALPINI (LEPIDOPTERA: NOCTUIDAE: CALPINAE) Introduction The most recent classification places Calpinae in the family Noctuidae (Goater et al. 2003, Kitching and Rawlins 1998, Fibiger and Lafontaine 2005, Lafontaine and Fibiger 2006, Mitchell et al. 2006). Presently, Calpin ae consists of four tribes: A nomini Grote 1882, Calpini Boisduval 1840, Phyllodini Hampson 1913, and Scoliopterygi ni Herrich-Schffer [1852] (Fibiger and Lafontaine 2005, Lafontaine and Fibiger 2006, Holloway 2005). Calpini are defined by the presence of socketted tearing hooks on the proboscis that are used for piercing the skin of fruits and mammals (Bnziger 1968, 1971, 1979a, 1982, Zaspel et al. 2007, Zaspel in press ). Calpini are cosmopolitan; however, many cal pine genera have geographic distributions that are more restricted (e.g., Gonodonta Hbner, Graphigona Walker, Calyptra Ochsenheimer). The tribe was recently catalogued by Fibiger and Lafontaine (2005) wherein they assigned genera to the tribe, suggesting Calpini consis ted of approximately 200 species in eleven genera. This publication focused on the Palearctic region and was therefore not inclusive of all genera comprising the tribe. In addi tion, the fruit-piercing genus Oraesia Guene, which has proboscis armature identical to that of Calyptra and Gonodonta, was excluded while seven genera lacking the diagnostic characteristics of the proboscis were included. The primary objective of the present checklist is to combin e the works of Holloway (2005) a nd Fibiger and Lafontaine (2005) into an updated checklist to complement re cent taxonomic studies (Bnziger 1983, Zilli and Hogenes 2002), a survey of calpine proboscis mo rphology (Zaspel et al. 2008), and phylogenetic research (Zaspel in preparation). This checklist also serves to correct minor taxonomic errors in the checklist of Poole (1989). 125

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All original descriptions for each genus, sp ecies, subspecies, and their synonyms have been studied. This checklist includes type localities if available, a complete references list, and corrections and changes to the nomenclature pres ented in the checklists of Poole (1989), Fibiger and Lafontaine (2005), and Holloway (2005). Author ship assignments for taxa described in the Lepidoptera Atlas of the Rei se der Novara follow Nssi g and Speidel (2007) and for convenience, are abbreviated In F.F.R. 1874. In summary, the following taxonomic changes are made: Culasta Moore 1881a is removed from synonymy from Calyptra Ochsenheimer (1816). This genus lacks the proboscis characters that define Calpini and is not consider ed a member; the tribal placement of Culasta remains undetermined. Eudocima talboti (Prout 1922) is placed in synonymy with Eudocima cajeta (Cramer 1775) and Graphigona antica Walker [1858] 1857b is placed in synonymy with G. regina (Guene In Boisduval and Guene 1852c). Four Eudocima species, E. behouneki, E. mazzeii, E. prolai, and E. treadawayi described by Zilli and Hoge nes (2002) are added to the checklist. Although Hilgartn er et al. (2007) suggest Hemiceratoides Strand 1911 is a member of the Calpini, this placement remains uncertain as species in the genus are tear feeders lacking proboscis features found in all othe r listed genera of Calpini. Phyllodes in tribe Phyllodini, has been associated with members of Calpini (e.g., Eudocima ) but also with Ophiusini (e.g., Miniodes ) (Holloway 2005). Given the previous associations of Phyllodes with both calpine and ophiusine genera, we recommend its continued placement in Phyllodini pending further morphological and molecular examination of addi tional species in the genus. Six genera: Africalpe Krger, Ferenta Walker, Gonodonta Hbner, Graphigona Walker, Oraesia Guene, and Tetrisia Walker, are added to Fibige r and Lafontaines (2005) ch ecklist based on characters of their proboscides. The following genera, previously treated as the tribe Calpini (sensu Fibiger 126

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and Lafontaine 2005), are removed based on their l ack of proboscides characters present in other members of this tribe, and categorized as genera whose tribal placement are as yet undetermined: Cecharismena Mschler, Goniapteryx Perty, Pharga Walker, Phyprosopus Grote, Psammathodoxa Dyar and Radara Walker. Checklist CALPINI AFRICALPE Krger, 1939: 348 Type-species: Africalpe intrusa Krger, by monotypy. intrusa Krger, 1939 ( Africalpe ) Libya nubifera (Hampson, 1907) ( Calpe) India vagabunda (Swinhoe, 1884) ( Oraesia ) Pakistan anubis (Rebel, 1947) ( Pseudocalpe ) Egypt anubia (Poole, 1989); misspelling CALYPTRA Ochsenheimer, 1816: 78 Type-species: Phalaena thalictri Borkhausen, by subsequent designation of Duponchel 1826: [3]. Calpe Treitschke, 1825 Hypocalpe Butler, 1883 Percalpe Berio, 1956 albivirgata (Hampson, 1926) ( Calpe ) China bicolor (Moore, 1883) ( Calpe) India canadensis (Bethune, 1865) ( Calpe ) Canada purpurascens (Walker, 1865) ( Plusiodonta ) USA sobria (Walker, 1865) ( Oraesia ) USA 127

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eustrigata (Hampson, 1926) ( Calpe ) Sri Lanka fasciata (Moore, 1882) ( Calpe) India labilis (Berio, 1970) ( Calpe) India fletcheri (Berio, 1956) ( Calpe) China gruesa (Draudt, 1950) ( Calpe) China hokkaida (Wileman, 1922) ( Calpe ) Japan hokkaido (Poole, 1989); misspelling hoenei (Berio, 1956) ( Calpe) China imperalis (Grnberg, 1910) ( Calpe ) India lata (Butler, 1881) ( Calpe ) Japan aureola (Graeser, 1889) ( Calpe) Russian Far East minuticornis (Guene In Boisduval and Guene, 1852a) ( Calpe ) Indonesia novaepommeraniae (Strand, 1919) ( Calpe) India nyei Bnziger, 1979b (Calyptra ) India ophideroides (Guene In Boisduval and Guene, 1852a) ( Calpe ) East Indies orthograpta (Butler, 1886) ( Calpe) India striata (Poujade, 1887) ( Calpe) China parva Bnziger, 1979b ( Calyptra ) India pseudobicolor Bnziger, 1979b ( Calyptra ) India subnubila (Prout, 1928) ( Calpe) Indonesia thalictri (Borkhausen, 1790) ( Phalaena ) Unknown centralitalica (Dannehl, 1925) ( Calpe ) Unknown pallida (Schwingenschuss, 1938) ( Calpe ) Turkey 128

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sodalis (Butler, 1878a) ( Calpe) Unknown EUDOCIMA Billberg, 1820: 85 Type-species: Phalaena salaminia Cramer, by monotypy. Elygea Billberg, 1820 Leptophara Billberg, 1820 Trissophaes Hbner, [1823] 1816 Othreis Hbner, [1823] 1816 Maenas Hbner, [1823] 1816 Rhytia Hbner, [1823] 1816 Acacallis Hbner, [1823] 1816 Ophideres Boisduval, 1832 Acacalis Agassiz, [1847] 1846 Ophioderes Agassiz, [1847] 1846 Othryis Agassiz, [1847] 1846 Khadira Moore, 1881b Adris Moore, 1881b Purbia Moore, 1881b Vandana Moore, 1881b Argadesa Moore, 1881b Halastus Butler, 1892 Eumaenas Tams, 1924 anguina (Schaus, 1911b) ( Trissophaes) Costa Rica apta (Walker, [1858] 1857b) ( Ophideres ) Brazil 129

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aurantia (Moore, 1877) ( Ophideres ) Andaman Islands rutilus (Moore, 1881b) ( Adris) Sri Lanka bathyglypta (Prout, 1928) (Othreis ) Indonesia behouneki Zilli and Hogenes, 2002 ( Eudocima ) Philippines boseae (Saalmller, 1880) ( Ophideres ) Madagascar caesar (Felder, 1861) ( Ophideres ) Indonesia cajeta (Cramer, 1775) ( Phalaena Noctua ) India multiscripta (Walker, [1858] 1857b) ( Ophideres) Sri Lanka talboti (Prout, 1922) ( Othreis ) Indonesia, NEW SYNONYMY cocalus (Cramer, 1777) ( Phalaena Noctua ) East Indies crepidolata (Lucas, 1894) ( Ophideres ) Australia maculata (Weber, 1801) ( Noctua) East Indies plana (Walker, [1858] 1857b) ( Ophideres ) Indonesia colubra (Schaus, 1911b) ( Trissophaes) Costa Rica discrepans (Walker, [1858] 1857b) ( Ophideres) Singapore archon (C. and R. Felder, In F.F.R. 1874) ( Ophideres ) Thailand dividens (Walker, [1858] 1857b) ( Ophideres ) Indonesia divitiosa (Walker, 1869b) (Ophideres ) Congo banakus (Pltz, 1880) ( Ophideres) Upper Guinea intricatus (Butler, 1892) ( Halastus ) Nigeria euryzona (Hampson, 1926) ( Khadira ) Madagascar felicia (Stoll, 1790) ( Phalaena Noctua ) Unknown formosa (Griveaud and Viette, 1960 ) (Khadira) Madagascar 130

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homaena (Hbner, [1823] 1816) ( Othreis) India ancilla (Cramer, 1777) ( Phalaena Noctua ) India bilineosa (Walker, [1858] 1857b) ( Ophideres) Sri Lanka collusoria (Cramer, 1777) ( Phalaena Noctua ) Unknown strigata (Donovan, 1804) ( Phalaena ) India hypermnestra (Cramer, 1780) ( Phalaena Noctua ) India imperator (Boisduval, 1833) ( Ophideres ) Madagascar iridescens (Lucas, 1894) ( Ophideres ) Australia pyrocrana (Turner, 1908) ( Ophideres ) Australia jordani (Holland, 1900) ( Ophideres ) Indonesia kinabaluensis (Feige, 1976) ( Othreis ) Borneo kuehni (Pagenstecher, 1886) ( Othreis ) New Guinea materna (Linnaeus, 1767) ( Phalaena Noctua ) Indiis chalcogramma (Walker, 1865) ( Ophideres ) Zambesi River hybrida (Fabricius, 1775) ( Noctua) India, Australia. mazzeii Zilli and Hogenes, 2002 ( Eudocima ) Philippines memorans (Walker, [1858] 1857b) ( Ophideres) West Coast of America mionopastea (Hampson, 1926) ( Othreis ) Malaysia muscigera (Butler, 1882) ( Purbia ) New Britain nigricilia (Prout, 1924) (Purbia ) Australia okurai (Okano, 1964) ( Adris ) East-Palearctic suthepensis Bnziger and Honey, 1984 ( Adris) Burma paulii (Robinson, 1968) ( Othreis ) Fiji 131

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phalonia (Linneaus, 1763) (Phalaena Noctua ) Africa dioscoreae (Fabricius, 1775) ( Noctua ) East Indies fullonia (Clerck, [1764] 1759) ( Phalaena ) Unknown obliterans (Walker, [1858] 1857b) ( Ophideres) Samoa pomona (Cramer, 1776) ( Phalaena Noctua ) India princeps (Boisduval, 1832) ( Ophideres ) New Guinea pratti (Bethune-Baker, 1906) ( Lagoptera ) New Guinea prattorum (Prout, 1922) ( Othreis ) Indonesia procus (Cramer, 1777) ( Phalaena Noctua ) Surinam columbina (Guene, In Boisduval and Guene 1852b) ( Ophideres ) Colombia scabellum (Guene, In Boisduval and Guene 1852b) ( Ophideres ) Unknown prolai Zilli and Hogenes, 2002 ( Eudocima ) Irian Jaya salaminia (Cramer, 1777) ( Phalaena Noctua ) China serpentifera (Walker, [1858] 1857b) ( Ophideres ) Dominican Republic sikhimensis (Butler, 1895) ( Adris ) Oriental abathyglypta (Prout, 1928) ( Othreis ) Indonesia smaragdipicta ( Walker, [1858] 1857b) ( Ophideres ) Borneo sultana (Snellen, 1886) ( Ophideres) Indonesia splendida (Yoshimoto, 1999) ( Othreis ) Oriental srivijayana (Bnziger, 1985) ( Othreis ) Oriental talboti (Prout, 1922) ( Othreis ) Wallacea toddi (Zayas, 1965) ( Othreis ) Cuba treadawayi Zilli and Hogenes, 2002 ( Eudocima ) Philippines 132

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tyrannus (Guene, In Boisduval and Guene 1852b) India amurensis (Staudinger, 1892) ( Ophideres ) Russian Far East FERENTA Walker, [1858] 1857a: 961 Type-species: Phalaena stolliana Stoll, by monotypy. cacica (Guene, In Boisduval and Guene 1852c) ( Ophideres ) Brazil castula (Dognin, 1912) ( Darceta ) Venezuela incaya Hampson, 1926 ( Ferenta ) Peru stollii (Hbner, [1823] 1816) ( Coronis ) stolliana (Stoll, In Cramer 1782) ( Phalaena Noctua ) Surinam GONODONTA Hbner, 1818: 11 Type-species: Gonodonta uncina Hbner, by subsequent designation of Grote 1902: 472. Revision: Todd 1959:1. Athysania Hbner, [1823] 1816 Dosa, Walker 1865 aequalis Walker, [1858] 1857a ( Gonodonta) Brazil aeratilinea Todd, 1973 ( Gonodonta) Peru amianta (Hampson 1924) ( Athysania ) Guyana biarmata Guene, In Boisduval and Guene 1852a ( Gonodonta) Brazil elegans Druce, In Godman and Salvin 1889 ( Gonodonta) Mexico evadens Walker, [1858] 1857a ( Gonodonta) Galapagos Islands galapagensis Todd, 1959 ( Gonodonta) Galapagos Islands bidens Geyer, 1832 ( Gonodonta) Cuba meridionalis Todd, 1959 ( Gonodonta) Brazil 133

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miranda Raymundo, 1908 ( Gonodonta) Brazil tenebrosa Todd, 1959 ( Gonodonta) Costa Rica chorinea (Cramer, 1782) ( Phalaena Noctua ) Surinam clotilda (Stoll, 1790) ( Phalaena Noctua ) Surinam clothilda (Poole, 1989); misspelling correcta Walker, [1858] 1857a ( Gonodonta) Mexico distincta Todd, 1959 ( Gonodonta) Venezuela ditissima Walker, 1858 ( Gonodonta) Brazil fernandezi Todd, 1959 ( Gonodonta) Guyana fulvangula Geyer, 1832 ( Gonodonta) Uruguay chrysotornus (Hampson, 1926) ( Athysania ) Guyana fulvidens Felder and Rogenhofer, In F.F.R 1874 ( Gonodonta) Colombia flavidens (Hampson, 1926) ( Athysania) Brazil holosericea Guene, In Boisduval and Guene 1852a ( Gonodonta) Colombia immacula Guene, In Boisduval and Guene 1852a ( Gonodonta) Fr. Guiana panoana (Schaus, 1933) ( Athysania ) Brazil incurva (Sepp, 1840) ( Phalaena ) Surinam dentata Felder and Rogenhofer, In F.F.R 1874 ( Gonodonta) Brazil elaborans Dyar, 1914 ( Gonodonta ) Dominica temperata Walker, [1858] 1857a ( Gonodonta) Venezuela teretimacula Guene, In Boisduval and Guene 1852a ( Gonodonta) Fr. Guiana velata Walker, [1858] 1857a ( Gonodonta) Unknown indentata (Hampson, 1926) ( Athysania ) Venezuela 134

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latimacula Guene, In Boisduval and Guene 1852a ( Gonodonta) Colombia lecha Schaus, 1911a ( Gonodonta) Costa Rica lincus (Cramer, 1775) ( Phalaena Bombyx ) Costa Rica superba Mschler, 1880 ( Gonodonta) Surinam maria Guene, In Boisduval and Guene 1852a ( Gonodonta) Brazil avangareza Schaus, 1911a ( Gonodonta) Costa Rica mexicana Schaus, 1901 ( Gonodonta) Mexico nitidimacula Guene, In Boisduval and Guene 1852a ( Gonodonta) Ile St. Thomas nutrix (Cramer, 1780) ( Phalaena Noctua ) Surinam acmeptera (Sepp, 1848) ( Phalaena) Surinam obsesa (Walker, [1865] 1864) ( Dosa ) Brazil camora (Felder and Rogenhofer, In F.F.R 1874) ( Canodia) Brazil paraequalis Todd, 1959 ( Gonodonta) Mexico parens Guene, In Boisduval and Guene 1852a ( Gonodonta) Guadeloupe plumbicincta Dyar, 1912 ( Gonodonta) Mexico primulina Druce, In Godman and Salvin 1887 ( Gonodonta) Guatemala pseudamianta Todd, 1959 ( Gonodonta) Venezuela pulverea Schaus, 1911a ( Gonodonta) Costa Rica pyrgo (Cramer, 1777) ( Phalaena Noctua ) Surinam serix Guene, In Boisduval and Guene 1852a ( Gonodonta) Colombia separans Walker, [1858] 1857a ( Gonodonta) Brazil sicheas (Cramer, 1777) ( Phalaena Noctua ) Surinam hesione (Drury, 1782) ( Phalaena Noctua ) Brazil 135

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uncina Hbner, 1818 ( Gonodonta) Brazil sinaldus Guene, In Boisduval and Guene1852a ( Gonodonta) Colombia sitia Schaus, 1911a ( Gonodonta) Costa Rica soror (Cramer,1780) ( Phalaena Noctua ) Surinam sphenostigma Todd, 1973 ( Gonodonta) Brazil syrna Guene In Boisduval and Guene1852a ( Gonodonta) Fr. Guiana unica Neumoegen, 1891 ( Gonodonta) USA uxor (Cramer, 1780) ( Phalaena Noctua ) Surinam marmorata Schaus, 1906 ( Gonodonta) Mexico walkeri Todd, 1959 ( Gonodonta) Costa Rica GRAPHIGONA Walker, [1858] 1857b: 1230 Type species: Ophideres regina Guene, by subsequent designation of Berio 1966: 59. regina (Guene In Boisduval and Guene 1852b) ( Ophideres ) Colombia antica Walker [1858] 1857b ( Graphigona) Brazil, NEW SYNONYMY gubernatrix (Guene In Boisduval and Guene 1852b) ( Ophideres ) Tropical America ORAESIA Guene, In Boisduval and Guene, 1852b: 362 Type-species: Noctua emarginata Fabricius, by subsequent designation of Warren In Seitz 1913. aeneofusa (Hampson, 1926) ( Calpe ) Panama albescens (Seitz, 1940) ( Calpe) Unknown argyrolampra (Hampson, 1926) ( Calpe) Colombia argyrosema (Hampson, 1926) ( Calpe ) Brazil argyrosigna ( Moore, [1884]) (Oraesia ) Sri Lanka basiplaga (Walker, 1865) ( Calpe) Dominican Republic 136

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camaguina Swinhoe, 1918 ( Oraesia ) Philippines cerne (Fawcett, 1916) (Calpe ) Ghana emarginata (Fabricius, 1794) ( Noctua ) India alliciens Walker, [1858] 1857a ( Oraesia ) India metallescens Guene, In Boisduval and Guene 1852a ( Oraesia ) Unknown tentans Walker, [1858] 1857a ( Oraesia ) India excavata (Butler, 1878a) ( Calpe) Japan excitans Walker, [1858] 1857a ( Oraesia ) Dominican Republic glaucocheila (Hampson, 1926) ( Calpe ) Brazil honesta Walker, [1858] 1857a ( Oraesia ) Dominican Republic igneceps (Hampson, 1926) ( Calpe) Br. Guyana nobilis Felder and Rogenhofer, In F.F.R 1874 ( Oraesia ) Brazil pierronii (Mabille, 1880) ( Odontina) Madagascar provocans Walker, [1858] 1857a ( Oraesia ) South Africa cuprea Saalmller, 1891 ( Oraesia ) Madagascar hartmanni Mschler, 1883 ( Oraesia ) South Africa rectistria Guene, In Boisduval and Guene 1852a ( Oraesia ) India serpens Schaus, 1898 ( Oraesia ) Mexico striolata Schaus, 1911a ( Oraesia ) Costa Rica stupenda Dognin, 1912 (Oraesia) Colombia subucula Dognin, 1910 ( Oraesia ) Paraguay triobliqua (Saalmller, 1880) ( Odontina) Madagascar wintgensi (Strand, 1909) ( Calpe) Rwanda 137

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PLUSIODONTA Guene, In Boisduval and Guene 1852b: 385 Type-species: Plusiodonta chalsytoides Guene, by subsequent designation of Desmarest In Chenu 1857: 123. Gadera Walker, [1858] 1857a Deva Walker, [1858] 1857a Odontina Guene, 1862 Tafalla Walker, 1869a Tinnodoa Nye, 1975 aborta Dognin, 1910 ( Plusiodonta) Colombia achalcea Hampson, 1926 ( Plusiodonta ) South Africa amado Barnes, 1907 ( Plusiodonta ) USA arctipennis Butler, 1886 ( Plusiodonta ) Australia auripicta Moore, 1882 ( Plusiodonta ) India basirhabdota Hampson, 1926 ( Plusiodonta ) Kenya casta (Butler, 1878b) ( Platydia ) Japan chalcomera Hampson, 1926 ( Plusiodonta ) Kenya clavifera (Walker, 1869a) ( Tafalla ) Honduras cobaltina Viette, 1956 ( Plusiodonta ) Madagascar coelonota (Kollar and Redtenbacher, 1844) (Plusia ) India agens (Felder and Rogenhofer, In F.F.R 1874) ( Plusia ) India chalsytoides Guene, In Boisduval and Guene 1852a ( Plusiodonta ) Indonesia conducens (Walker, [1858] 1857a) ( Deva ) Sri Lanka commoda Walker, 1865 ( Plusiodonta ) Sierra Leone 138

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compressipalpis Guene, In Boisduval and Guene 1852a ( Plusiodonta ) N. America insignis Walker, 1865 ( Plusiodonta) USA suffusa Hill, 1924 ( Plusiodonta ) USA cupristria Kaye, 1922 ( Plusiodonta) Trinidad dimorpha Robinson, 1975 ( Plusiodonta ) Fiji effulgens Edwards, 1884 ( Plusiodonta ) Mexico euchalcia Hampson, 1926 ( Plusiodonta ) Malawi excavata (Guene, 1862) ( Odontina ) Unknown gueneei (Viette, 1968) ( Odontina) Madagascar incitans ( Walker, [1858] 1857a) ( Gadera ) Unknown ionochrota Hampson, 1926 ( Plusiodonta ) Ghana macra Hampson, 1926 ( Plusiodonta ) Kenya malagassy (Viette, 1968) ( Odontina ) Madagascar megista Hampson, 1926 ( Plusiodonta ) Kenya miranda Schaus, 1911a ( Plusiodonta ) Costa Rica multicolora (Bethune-Baker, 1906) ( Deva ) New Guinea natalensis Walker, 1865 ( Plusiodonta ) South Africa detracta Walker, 1865 ( Plusiodonta ) South Africa nummaria Felder and Rogenhofer, In F.F.R 1874 ( Plusiodonta ) South Africa tripartita Walker, 1865 ( Plusiodonta ) South Africa nictites Hampson, 1902 ( Plusiodonta ) South Africa nitissima Schaus, 1911a ( Plusiodonta ) Costa Rica repellens (Walker, [1858] 1857a) ( Gadera ) Unknown 139

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speciosissima (Holland, 1894) ( Deva ) Cameroon stimulans (Walker, [1858] 1857a) ( Deva ) Dominican Republic theresae Holloway, 1979 ( Plusiodonta ) New Caledonia thomae Guene, In Boisduval and Guene 1852a ( Plusiodonta ) Virgin Islands tripuncta (Bethune-Baker,1906) ( Marcipa ) New Guinea wahlbergi (Felder and Rogenhofer, In F.F.R 1874) ( Plusia ) South Africa africana (Holland, 1894) ( Deva ) Gabon regina (Guene, In Boisduval and Guene 1852b) ( Ophideres ) Colombia TETRISIA Walker, 1867: 186 Type species: Tetrisia florigera Walker, by monotypy. florigera Walker, 1867 ( Tetrisia ) Colombia magnifica (Schaus, 1911b) ( Graphigona) Costa Rica roseifer (Felder and Rogenhofer, In F.F.R 1874) ( Graphigona ) Brazil Genus and tribal placement undetermined 1. Cecharismena Mschler, 1890 Checharismena, Fibiger and Lafontaine 2005; misspelling 2. Culasta Moore, 1881a 3. Goniapteryx Perty, In Spix 1833 4. Hemiceratoides, Strand 1911 5. Hypsoropha Hbner, 1818 6. Pharga Walker, 1863 7. Phyprosopus Grote, 1872 8. Psammathodoxa Dyar 1921 9. Radara Walker, 1862 140

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CHAPTER 5 ANOTHER BLOOD FEEDER? EXPERIMENT AL FEEDING OF A FRUIT-PIERCING MOTH SPECIES ON HUMAN BLOOD IN TH E PRIMORYE TERRITORY OF FAR EASTERN RUSSIA (LEPIDOPTE RA: NOCTUIDAE: CALPINAE) Introduction The genus Calyptra Ochsenheimer (Lepidoptera: Noctui dae: Calpini) includes what are commonly known as vampire moths, so named because of their ability to pierce mammalian flesh and feed on blood. These are medium sized moths, with wingspans ranging from 35-72 mm in size (Bnziger 1983, Table 1) (Figs. 5-1, 52, 5-3). Species in th is genus occur in S. Europe, eastern Africa, sub-Himalayan regions of S. Asia, the Manchurian subregion, and are broadly distributed th roughout S.E. Asia. Calyptra species have enjoyed popularity among members of the entomological community due to their modified proboscides equipped with strongly sclerotized barbed hooks used for pierci ng through both thick and hard skinned fruits such as peaches, plums, and citrus as well as mammals (Bnziger 1982, Zaspel pers. obs., Fig. 54). Of the 17 known Calyptra species (Bnziger 1983), C. eustrigata (Hampson), C. minuticornis minuticornis (Guene), C. orthograpta (Butler), C. bicolor (Moore), C. fasciata (Moore), C. ophideroides (Guene), C. parva Bnziger and C. pseudobicolor Bnziger have been reported to pierce mammalian skin, the latte r five also of man, und er natural conditions while C. fletcheri (Berio) has done so in experiments (Bnziger 1968, Bnziger 1989). These species are considered facultative or opportunistic blood-feeders primarily in subtropical areas in southern Asia and tropical Southeast Asian count ries: their hosts are typically ungulates such as cattle, tapirs, zebu, and occasionally elephants and humans; female Calyptra adults have not been documented feeding on blood (Bnziger 1989b). At least four additional closely related genera ( Eudocima Gonodonta, Oraesia and Plusiodonta ) have apparently homologous 141

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proboscides modifications used fo r fruit-piercing, but the occurre nce of blood-feeding in those species has not been observed (Bnziger 1979 and personal communication Zaspel, unpublished data ). Although it is known that some fruit-piercing moths are of great economic importance, e.g., Eudocima fullonia and to a lesser degree even some Calyptra spp. (Fay 2002, Fay and Halfpaff 2006, Sands 1993, Todd 1959, Yoon and Lee 1974) their potential as vectors of human or animal disease remains a possibility, however whether a real danger of vectoring disease exists is unknown (Bnziger 1980, Bnziger 1989). The purpose of this paper is to document the first case of Calyptra feeding on human blood in Far East ern Russia and the novel finding of blood feeding by the species C. thalictri under experimental conditions. Materials and Methods Description of observation sites During an expedition in the Primorye Territory of Far Eastern Russia (Fig. 5-5) in July, 2006, I sought to observe feed ing behaviors of the three Calyptra species ( C. hokkaida Wileman, C. lata Butler, and C. thalictri Borkhausen) in this region (Kononenko 1990a; Remm 1980b). None of these species had been recorded feed ing on mammalian blood and have been considered exclusive fruit piercers. Since the complete geographic occurrenceand precise phylogenetic originsof blood feeding Calyptra remain unknown, an objective of this work was to determine whether Calyptra species that occupy the northern exte nt of their range were hematophagous. The southern part of the Russian Far East (Primorye territory) lies in a zone of Manchurian coniferous and mixed c oniferous and broad leaved forests with very rich and diverse vegetation. The main forest formations in the region are AbiesPicea taiga in the upper and mid mountain belts, Pinus koraiensis mixed forest in the mid mountain belt, Abies nephrolepsis mixed forest in the south of the region, deciduous broad leaved forest and forest dominated by Quercus mongolica (Kurentzov 1965, Richter 1961). The mixed and deciduous forests of the 142

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south Far East contain many east Asian trees and sh rubs that are absent from Siberia, such as Quercus Fraxinus Acer Tilia Ulmus Carpinus Phellodendron, Maakia Aralia Calopanax, Actinidia Schisandra (Kurentzov 1965, Richter 1961). The sub-alpine, and mountain tundra belts are fragmentary, and occur in the Sikhote-Alin mountain range above 1500m in the central and northern part of the region. The foreststeppe zone occurs mainly in the southwestern portions of the region (Kurentzov 1965, Richter 1961). The climate of the Primorye territory is dominated in part by monsoon features. The annual temperature in Primorye, is + 4 C, temper ature in January: 10-15C, in July it is over +20 C (Kurentzov 1965, Richter 1961). The an nual precipitation is 800 mm, roughly 70 75% of which falls from July to September. Northwesterly winds dominate in winter, while southeast winds prevail in the summer (Kurentzov 1965, Richter 1961). We collected Calyptra specimens at two primary sites (d esignated Sites 1 and 2; Figs. 56, 5-7, 5-8, 5-9) and three subsites (designate d subsites 1a and 1b; subsites 2a, 2b, and 2c respectively). Collecting site 1 (including subsites 1a and 1b) was in the vicinity of the Kraunouka Village at the Borisovskoe Hunting Area, roughly 20 km west of Ussuriisk, Russia (Figs. 5-6 and 5-8: N 43 44.577, E 131 38.218; 287 ft.). This is a popular hunting site for wild boar (Sus scrofa sp.), two species of deer ( Cervus nippon and Capreolus capreolus), and bear ( Ursus thibetanus ), situated in the easternmost spurs of the East Manchurian montane system (Fig. 5-6). Although it has not yet been observed in this region, these mammals could potentially serve as hosts of adult Calyptra species. This area contains low elevation mountains (100-300 m), hills and cliffs in the upper reaches of the Kraunouka River. The vegetation around the collecting sites consists primarily of broad leaf forests dominated by oak ( Quercus mongolica ) (Kurentzov 1965, Richter 1961). The vegetation along the river valley is considerably more 143

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diverse broad-leafed forest with variously dominant species such as Juglans mandshurica Ulmus japonica and, Vitis amurensis Mixed broad-leaf coniferous forest with coniferous trees such as: Abies holophila A. nephrolepsis Pinus koraiensis and P. funebris cover the upper reach of Kraunouka River (Kurentzov 1965, Richter 1961). Most relevant, are the pres ence of larval food plants of the local species of Calyptra : Menispermum dahuricum, Thalictrum contortum T. simplex, and T. amurensis are abundant in meadows (K urentzov 1965, Richter 1961). The second collecting site (subs ites 2a, 2b, and 2c) was at th e Gornotayeznaya Biological Station Far Eastern Branch of the Russian Academy of Sciences (20 km east of Ussurisk). This collecting site was in the vicinity of Gornotayez noe village situated in southwestern spurs of Sikhote-Alin montane range (Figs. 5-7 and 5-9: N 43 41.917 E 132 09.131). The area is in the vicinity of low elevation mount ains (200-400 m). The vegetation around the collecting site is similar to first collecting locality and consists of broad-leaf forest in a creek valley. Wild rosaceous plants are less well represented here th an in the former site; however, apple, pear, cherry and apricot trees are cultivated in gardens. The fruits of Rosaceae are commonly attacked by many fruit-piercing mo ths, including some Calyptra spp. (Bnziger 1 971, Bnziger 1982). The southern slopes of the hills are covered by secondary forest dominated by Quercus mongolica and Corylus mandschurica in the understory; the northern slopes are covered by mixed broad leaf coniferous fo rest with native and planted trees and pure primary forest dominated Pinus koraiensis situated in the upper reaches of the Krivoi kljuch creek (Kurentzov 1965, Richter 1961), roughly 10 km from the collecting site. Experimental methods Moths were collected using standard techniques: suspended white sheets illuminated by a 60 watt mercury vapor lamp (HgVpr, Winter 2000). Live specimens were collected into separate 10 dram plastic vials. Upon return to the field station at primary collec ting sites 1 and 2, male 144

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specimens were retained alive overnight. Fema les were transferred into 95% EtOH and males were placed in a small rearing te nt with small pieces of wet cotton for about four hours. Each specimen was carefully removed from the tent a nd placed in a numbered live vial (Table 5-1). Feeding trials were conducted to determine whic h, if any, of the additi onal specimens collected would penetrate human skin and feed on blood. The moths were presente d with two separate feeding opportunities conducted over a 24-hour period. The first tr ial was always the morning after the moths were collected, and started between 0800h and 1000h second trial began in the evening between 1900h and 2100h. The trials were conducted by inserting a thumb into the vial in such as way so that the moth could not escape, and each moths behavior was recorded for ten minutes. If the moth began to feed, or made an attempt to feed beyond the ten minute time period it was not interrupted. If a moth did take a blood meal, it was eliminated from the experiment and recorded as a positive blood-feeder. This was done to minimize potential injury and/or allergic reactions on the part of the subjects. If no feeding activity was observed after ten minutes, or if previous feeding behavior(s) ceas ed, the thumb was removed from the vial and the moth was given another opportunity to feed durin g trial 2. All species identifications were confirmed based on external mor phology and genitalic dissections. Results On July 14 th 2006 a male specimen of Calyptra thalictri (Fig. 5-1) was collected in Kraunouka about 2 km from the field station in the hunting area (subsite 1a) at approximately 2300h. This specimen was placed in a plastic live vial (designated vial #1, Table 5-1) and a human thumb inserted into the vi al to observe potential piercing behavior. After three minutes, the moth inserted its proboscis into the thumb just below the nail and began to uptake blood for approximately 2 minutes. Three additional spec imens of this species (two males, and one female) were also collected the same night at subsite 1a. The following morning (7-15-06: 145

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1047h), Specimen in vial #1 was given another oppor tunity to feed on human blood (Table 5-1). During this time, the moth grazed its proboscis ac ross the subjects thumb, lapping moisture off of the skin. After approximately five minutes, th e moth inserted the tip of its proboscis into a crease in the skin on the joint of the thumb and pi erced through the skin (Fig. 5-10A). The moth sucked blood briefly (less than 30 seconds) and without removing the tip of the proboscis pierced further into the wound and then continued sucking blood. This behavior (intermittent piercing followed by feeding) continued for six minutes before the moth was removed, and the wound examined (Fig. 5-10B). The same methodology wa s used to determine whether the additional male specimens would feed. However, the specimens only licked the skin, presumably unsuccessful attempts at piercing. All specimens were ultimately placed in 95% EtOH for future molecular study. On July 15 th at approximately 2030h (dusk) a male C. thalictri was captured outside the perimeter of the field station in the Borisovskoe Hunting Area. This specimen was resting on the trunk of a cherry shrub when it was hand collected. The specimen slowly crawled outside the slightly compressed hand through the opening betw een the forefinger and thumb, where it rested. The moth uncoiled its proboscis several times, touc hing the skin below the thumbnail. The moth made several attempts to pierce the skin with its proboscis, finally settling on a crease in the skin area 7-8 mm above the border of the thumbnail (the same location as the previous blood-feeding specimen). The moths proboscis slowly pe netrated the skin. As with the first Calyptra thalictri specimen collected (specimen in vial #1, Table 51), this moth (vial #4, Table 5-1) pulled its proboscis slightly out of the wound, and then pi erced further into the wound. When the moth pulled its proboscis out of from the wound, it wa s saturated with blood (Figs. 5-10C, 5-10D, 510E). This feeding continued for an additional seven minutes. This incident of blood feeding 146

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should be considered semi-natural, as the moth was not enclosed in a vial, yet did not actively seek out the host and could have been disoriented given the time of day was earlier than its typical flight time. The subject observed sl ight swelling in the area around the wound, and described a feeling of slight stinging pain fo r 2-3 hours following the a ttack. After feeding ceased, the specimen was placed in a vial and ke pt alive overnight. At 0130h the live vial containing this moth had several large drops of blood at the bottom indi cating excretion of the blood meal (Fig. 5-10F). Later that morning all blood droplets we re absent, suggesting re-uptake of the blood meal, and when the moth was remove d from the vial and tr ansferred into EtOH, additional blood droplets were excreted from the tip of the abdomen. Between the hours of 2230h and 0100h on 15 July, thirteen additional Calyptra specimens were collected at a third site about 10 km from the hunting ar ea (subsite 1b) using the same methods. Of these specimens, eight were C. thalictri (Fig. 5-1; three females and five males), and five were C. lata (Fig. 5-2; one female and four males). All specimens were collected into separate live vials and were transported to the Gor notayeznaya Biological Station. At the Gornotayeznaya Biological Station, the females were given the opportunity to feed, but made no attempts to do so. The lack of attempts to feed or even lick moisture from the subjects thumb corroborates previously publi shed work that states female Calyptra species do not feed on blood (Bnziger 1989 [b]). Females were not included in future feeding trials. In addition to the two blood feeder s mentioned above, one of the male C. thalictri specimen collected from subsite 1b (vial #5, Table 5-1) pierced the senior authors thumb and fed on blood for four minutes and th irty seconds. The remaining C. thalictri specimens only attempted to pierce but uptook moisture from the skin. The C. lata specimens made no attempt to feed. 147

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During the course of these feed ing experiments, seven additional Calyptra thalictri specimens, and six specimens of C. lata were collected in the vicinity of the Gornotayeznaya Biological Station and subjected to feeding tria ls. These specimens were presented the same feeding opportunities those specimens taken from the Borisovskoe Hunting Area. Of the C. thalictri collected at various sites surrounding the Go rnotayeznaya Biologic al Station, none fed on blood and only one specimen made a weak atte mpt to pierce the skin (but did not draw blood); all specimens, however, did graze the skin w ith the proboscis, lapping moisture off of the thumb. No attempts to pierce or lap moisture from skin were observed by any of the C. lata specimens. Sixteen additional specimens of C. lata and C. thalictri were collected on 21 July and were either pinned or transferred into tu bes with 95% EtOH for future morphological and molecular study. Of the nine male C. thalictri specimens collected at Primary site 1 (and subsites therein), 44% were successful at piercing skin and feeding on blood. Calyptra lata specimens from Primary site 1 did not attempt to pierce. One attempt to pierce was made by a C. lata specimen from Primary site 2; C. thalictri specimens from Primary site 2 made no attempts to pierce. A summary of feeding behaviors for the Calyptra specimens subjected to feeding trials during 14-15 July and 17-20 July is provided in Table 5-1. Discussion These are the first recorded experime ntal observations of blood feeding by a Calyptra species in the Primorye Territory. Although und er experimental and se mi-natural conditions, they represent the first documented occurrence of blood feeding by Calyptra in a temperate region, and a new blood-feeding species record for the genus. The observed blood feeding behavior of C. thalictri specimens in this study consisted of spindle movements, head oscillations, followed by frequent partial withdrawl and re-inserti ons of the proboscis; thus, the blood feeding behavior of C. thalictri under the conditions reported in this paper are identical to 148

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those previously described for the blood feeding species C. eustrigata (Bnziger 1968, Bnziger 1980). It was unclear from the feeding experime nts whether saliva was re leased or if blood was regurgitated during feeding. Although indistinguishable by ge nitalia and proboscis morphology (including development of armrature), C. thalictri specimens collected in the Primorye Territory vary phenotypically from those that occur in Palearctic regions: specimens collected in Primorye have dark green forewings as opposed to the red-orange colored Palearctic specimens (compare Figs. 5-1 and 5-11). It is possibl e that the two populations repres ent two different species, but addressing this question is beyond the scope of this paper. The fact that Calyptra thalictri specimens from the second collectin g site did not pierce skin and feed on blood is also consistent with previous published feeding behavior reco rds for this species (Bnziger 1970, Bnziger 1971). Similar differential feeding behaviors have also been reported in other Calyptra species, e.g., C. fasciata (Bnziger 1989 [b]). Whether or not Calyptra thalictri are feeding on blood under natural conditions in the Primorye Region is presently under furthe r investigation. It is possible that C. thalictri specimens feeding behavior was different in the enclosed environmen t as opposed to their behavior under completely natu ral conditions; however, these pr eliminary observations indicate that C. thalictri can be induced to pierce human skin to suck under experimental conditions, while C. lata cannot. These findings are also consiste nt with previous studies reporting the blood feeding behavior in these moth s is restricted to the males. It has been suggested that males may engage in zoophilous feeding behaviors as a re sult of sugar, salt, or amino acid deficiency (Scoble 1992). When the fruit or nectar hos ts supplying these nutri ents are unavailable, butterflies and moths will seek alternate substrates on which to feed (puddles, dung, urine, sweat, 149

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150 tears, and blood). Bnziger (1972, 1990) de monstrated that the eulachryphagous moth Lobocraspis griseifusa Hampson has proteinases with which it can digest protein contents in the tears of mammals while such hemilachryphagous pyralids as Filodes mirificalis Lederer are, like the very vast majority of adu lt Lepidoptera, incapable of prot ein digestion. It is uncertain whether such a mechanism exists in blood feeding Calyptra spp. It should be noted however, that the skin piercing behavior followed by su cking blood from the mammalian host is derived from the fruit piercing habit, as opposed to othe r zoophilous feeding behavi ors, e.g., tear feeding. No full analysis of the digestive capabilities of Calyptra has yet been published; however, the observations in this study support previous work that suggests blood-feeding moths may engage in this behavior facultatively, depending on regional availability of mammalian versus vegetative hosts.

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Table 5-1. Summary of feeding be haviors for moth specimens collected in Primorye Terriotry of Far Eastern Russia from July 14t 15 th 2006 and July 17-20 th 2006. LM = Licking moisture off skin with pr oboscis, PA = Piercing attempted, PBF = Piercing and blood-feeding, NA = No attempt to lick moisture, pier ce skin, or feed on blood, DT = Dead at time of trial. Species Site Sex Date Collected Vial Number Feeding Trial Behavior Time C. thalictri Subsite 1a M 7-14-06 1 1 PBF 2 min. 2 PBF 6 min. C. thalictri Subsite 1a M 7-14-06 2 1 LM 10 min. 2 LM 10 min. C. thalictri Subsite 1a M 7-14-06 3 1 LM 10 min. 2 LM 10 min. C. thalictri Primary 1 M 7-15-06 4 1 PBF 11 min. C. thalictri Subsite 1b M 7-15-06 5 1 LM 10 min. 2 PBF 4.5 min. C. thalictri Subsite 1b M 7-15-06 6 1 LM 10 min. 2 LM 10 min. C. thalictri Subsite 1b M 7-15-06 7 1 LM 10 min. 2 LM 10 min. 151 C. thalictri Subsite 1b M 7-15-06 8 1 LM 10 min. 2 LM 10 min. C. thalictri Subsite 1b M 7-15-06 9 1 LM 10 min. 2 LM 10 min. C. lata Subsite 1b M 7-15-06 10 1 NA 10 min. 2 NA 10 min. C. lata Subsite 1b M 7-15-06 11 1 NA 10 min. 2 NA 10 min. C. lata Subsite 1b M 7-15-06 12 1 NA 10 min. 2 NA 10 min. C. lata Subsite 2a M 7-17-06 13 1 LM 10 min. 2 PA 10 min. C. thalictri Subsite 2b M 7-18-06 14 1 LM 10 min. 2 LM 10 min.

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Table 5-1. Continued Species Site Sex Date Collected Vial Number Feeding Trial Behavior Time C. thalictri Subsite 2b M 7-18-06 15 1 LM 10 min. 2 LM 10 min. C. thalictri Subsite 2b M 7-18-06 16 1 LM 10 min. 2 LM 10 min. C. thalictri Subsite 2b M 7-18-06 17 1 LM 10 min. 2 LM 10 min. C. thalictri Subsite 2b M 7-18-06 18 1 NA 10 min. 2 NA 10 min. C. thalictri Subsite 2b M 7-20-06 19 1 LM 10 min. 2 DT n/a C. thalictri Subsite 2b M 7-20-06 20 1 LM 10 min. 2 NA 10 min. C. lata Subsite 2b M 7-20-06 21 1 NA 10 min. 2 NA 10 min. C. lata Subsite 2c M 7-21-06 22 1 NA 10 min. 2 NA 10 min. C. lata Subsite 2c M 7-21-06 23 1 NA 10 min. 2 NA 10 min. C. lata Subsite 2c M 7-21-06 24 1 NA 10 min. 2 NA 10 min. C. lata Subsite 2c M 7-21-06 25 1 NA 10 min. 2 NA 10 min. C. lata Subsite 2c M 7-21-06 26 1 NA 10 min. 2 NA 10 min. 152

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Figure 5-1. Adult habitus image. Calyptra thalictri Male. 153

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Figure 5-2. Adult habitus image. Calyptra lata Male. 154

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Figure 5-3. Adult habitus image. Calyptra hokkaida, Male. 155

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TH 4 Figure 5-4. Proboscis of Calyptra thalictri : TH = Tearing Hooks. 156

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Figure 5-5. Map of Primorye Re gion of Far Eastern Russia. 157

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Figure 5-6. Primary collecting site 1. 158

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Figure 5-7. Primary collecting site 2. 159

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Figure 5-8. Map of primary collecting site 1. 160

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Figure 5-9. Map of primary collecting site 2. 161

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Figure 5-10. (A) Image of Calyptra thalictri feeding on human thumb (JMZ); (B) Image of subjects wound (JMZ) after piercing and feeding by C. thalictri; (C) Image of Calyptra thalictri feeding on human thumb (VK); (D) Image of Calyptra thalictri feeding on human thumb (VK); (E) Image of Calyptra thalictri feeding on human thumb; (F) Live vial showing blood droplets excreted by C. thalictri 162

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Figure 5-10. Calyptra thalictri feeding on raspberry during ni ght observations (under natural conditions) in S. Europe, photo by Hans Bnziger. 163

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CHAPTER 6 MICROBIAL DIVERSITY ASSOCIATED WITH THE FRUIT-PIERCING AND BLOODFEEDING MOTH CALYPTRA THALICTRI (LEPIDOPTERA: NOCTUIDAE: CALPINAE) Introduction Although blood feeding is common in many inse ct orders, within the Lepidoptera skin piercing and blood feeding are re stricted to the moth genus Calyptra Calyptra Ochsenheimer (Lepidoptera: Noctuidae: Calpini) is comprised of both obligator y fruit-piercing and facultative blood-feeding species. Of the 17 Calyptra species (Bnziger 1983), half have males that have been documented to feed on blood under either na tural or experimental conditions (Bnziger 1968, Bnziger 1989). Males are opportunistic blood-feeders pr imarily in subtropical and tropical areas in Asia; their hosts are typica lly ungulates such as cattle, tapirs, zebu and, occasionally, elephants and humans. It has been shown that blood-feedi ng males sequester up to 95% of the NaCl from the blood (Bnziger 2007). Calyptra females have not been shown to feed on blood (Bnziger 1989) and it is unclear whether males provide substances from the ingested blood to them during mating (Bnziger 2007). The remaining non-blood feeding males of Calyptra species are thought to be obligatory piercers of fruits such as peaches, pl ums, and citrus (Bnziger 1982). However, males of C. thalictri thought to be limited to fruit piercing, were shown to pierce human skin and feed on a human host for up to 10 min (Zaspel et al. 2 007). It is unknown whet her these moths ingest pathogens or parasites during the blood meal; however, if present, the survival of such disease microorganisms in Calyptra species could be dependent on th e presence of other microorganisms in the midgut (Azambuja et al. 2005). Recent findi ngs suggest that microbial gut endosymbionts may affect the ability of an insect vector to transmit disease agents to their vertebrate host(s) (St. Andr et al. 2002). Thus, the potentia l of these moths to vector disease may be related to their microbial gut fauna. 164

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It has been speculate d that blood-feeding Calyptra males are vectors of human or animal disease, but whether a real danger exists is unknown (Bnzi ger 1980, Bnziger 1989), and the occurrence of this C. thalictri population (and other species) in remote areas has hindered study. An important step in understan ding the biology and feeding behavior of the fruit-piercing and blood-feeding meals of Calyptra will be the identification of their associated microbial community. The goals of this study were to conduct a survey for microorganisms associated with male specimens of C. thalictri obtained in Far Eastern Russia. Males of these moths were surveyed for Archaea, Eubacteria, fungi includi ng yeast-like organisms, Microsporidia, and Wolbachia using a high-fidelity PCR assay. Followi ng terminology used in recent bacterial surveys of Lepidoptera, and because bacterial species are difficult to delineate, organisms detected in male C. thalictri in this study are refered to as p hylotypes (Broderick et al. 2004). Additionally, in an effort to understand which, if any, were co nsistently associated with C thalictri males, the proportion of individuals that contained each phylotype is reported. Materials and Methods Specimens Nine males of C. thalictri were collected at two sites in July 2006 in the Primorye Territory in Far Eastern Russia. Collecting site one was in the vi cinity of the Kraunouka Village at the Borisovskoe Hunting Area, roughly 20 km west of Ussuriisk, Russia (N 43 44.577, E 131 38.218; 84.5 m). The second site was at the Go rnotayeznaya Biological Station Far Eastern Branch of the Russian Academy of Sciences (20 km east of Ussurisk). This collecting site was in the vicinity of Gornotayeznoe village situat ed in southwestern spurs of the Sikhote-Alin montane range (N 43 41.917 E 132 09.131). Moths were collected using suspended white sheets illuminated by a 60-W mercury vapor lamp (HgVpr, Winter 2000). Live specimens were 165

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collected into separate 10-dram plastic vials. Specimens were transferred into 95% EtOH and transported to the United States where they we re stored separately at -80C prior to DNA extraction. Surface Sterilization A sterilization method to remove the DNA of microorganisms associated with the external surfaces of C. thalictri was used because it has been demonstrated to both kill surfaceinhabiting microbes and to eliminate their DNA when other solutions fail to do so consistently. This protocol does not interfer e with PCR analysis of gut or reproductive tr act endosymbionts (Meyer 2007, Meyer and Hoy in press ). Whole bodies of individual moths each were placed in a 15-mL plastic centrifuge tube f illed with 6% sodium hypochlorit e solution for 3 min and then rinsed five times with autoclaved double de-i onized water using sterile filtered pipette tips (Posada and Vega 2005). The abdomens then were removed from each moth using sterilized spring scissors and sterilized fine-tip forceps and placed in separate, covere d sterile petri plates. Spring-scissors, cleaned with 6% sodium hypochlorite solution followed by 100% EtOH, and held in a flame for 5 sec, were used to make a small incision at the tip of the abdomens of C. thalictri males under a dissecting microscope to exam ine the genitalia in order to confirm the identity of each specimen. All abdominal contents, including the digestive and reproductive tracts, hemolymph, and fat body were removed and placed in sterile 1.5-mL sterile centrifuge tubes. In order to determine whether the mo ths had specific microbes associated with the salivary glands and/or their mouthpa rts, heads with the proboscis attached and the contents of the pharynx were removed from each specimen after th e bleach treatment using a sterilized fine-tip forceps under a dissecting microscope Heads and associated mouthpa rts were rinsed with sterile water in the same manner as the abdomens and th en placed in sterile 1.5 -mL centrifuge tubes. Head and gut contents were stored at -80C prior to DNA extraction. These procedures were 166

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conducted in a different room from where th e DNA extractions and PCR reactions were conducted. DNA Extraction Individual surface-sterilized moth abdomens or heads were homogenized with a disposable blunt-ended sterile pi pette tip for 4 min each. Homogenized tissues were used for DNA extraction using PUREGENE reagents (Gentr a Systems, Minneapolis, MN) according to the manufacturers protocol. Due to the large amounts of tissue and fat body in the moths, two protein precipitations were performed on all abdo minal samples. DNA pellets were dried for 20 min and re-suspended in 100-L of sterile water. To further purify the samples, they were subjected to a chloroform extraction: 200-L of sterile water was added to 200-L of chloroform and added to the samples; the samples were then mixed by hand with a disposable sterile blunt pipette tip until smooth, and then placed on ice for 5 min. Samples were centrifuged for 15 min at 12,000 rpm and the supernatant was removed and transferred into a clean 1.5-mL centrifuge tube. DNA was precipitated using isopropanol at -80C. Samples were centrifuged for 15 min at 12,000 rpm and the DNA pellets were washed with 70% EtOH, air dried for 5 min, and resuspended in 100-L of sterile water. In or der to prevent contaminati on of the surface-sterilized samples, all DNA extractions were performed in an area separated from where the high-fidelity PCR was conducted. The quantity of DNA in each sample wa s determined using a spectrophotometer (BIORAD SmartSpec Plus, Hercules, CA). For the initial survey, 1-L of DNA from each abdominal or head extract was pooled into a single 1.5-mL microc entrifuge tube and 1-L of this pooled sample was used in PCR analyses with the primers lis ted in Table 6-1A. These samples were pooled to reduce the chance of missing potential microbial asso ciates that were not present in 100% of the population. In order to increa se the amount of DNA available for subsequent 167

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phylotype-specific PCRs on individual specimens, a multiple displacement amplification (MDA) reaction was performed on 1-L of DNA of each extract following the recommendations of the manufacturer (Dean et al. 2001, Dean et al 2002, Jeyaprakash a nd Hoy 2004); resulting Genomphi amplification products (Amersham Bioscien ces Piscataway, NJ) were stored at -80C. This supply provided us with additional DNA in the event that a large number of microbial endosymbionts were found in the pooled DNA. High Fidelity Polymerase Chain Reaction A 25-L high-fidelity PCR that contained 50 mM Tris, pH 9.2, 16 mM ammonium sulfate, 1.75 mM MgCl 2 350 mM dNTPs, 800 pmol primers, 1 unit Pwo DNA polymerase and five units of Taq DNA polymerase (Roche Molecular Bioc hemicals, Indianapolis, IN) was used to amplify 1 L of template DNA with the PCR react ion conditions listed in Hoy and Jeyaprakash (2005) with the primers listed in Tabl e 6-1A. Three linked profiles were used (i) 1 cycle of denaturation at 94C for 2 min; (ii) 10 cycles of denatu ration at 94C for 10 s, annealing at 65C for 30 s, and elongation at 68C for 1 min; and (iii) 25 cy cles of denaturation at 94C for 10 s, annealing at 65C for 30 s, an elongation at 68C for 1 min plus an additional 20 s for each consecutive cycle. Agarose gel electrophoresis (1% TAE gels) was used to separate PCRamplified DNA, which was stained with ethidium bromide and visualized with ultraviolet light. Cloning and Restriction Fragment Length Polymorphism Analysis PCR products were purified with the QI Aquick PCR Purification Kit (QIAGEN, Valencia, CA). To enhance cloning efficiency, 10 L of all PCR products were combined with 1-L dATP, 1-L Taq DNA polymerase, 1-L buffer, and then placed in the thermocycler at 72C for 1 h. PCR products we re then cloned into pCR2.1 TO PO following the manufacturers protocol (Invitrogen, Carlsbad, CA); colonies were grown on petri plat es at 37C overnight. Individual E. coli transformants were selected at random, grown overnight in 5-mL of LB broth 168

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+ 20 L of ampicillin, and plasmid DNA was extr acted using QIAGEN Pl asmid Mini Columns (QIAGEN, Valencia, CA). The presence and size of inserted DNA was confirmed by gel electrophoresis of plasmids following digestion with Eco R I for 2 h at 37C. Rsa I digests were used for the restriction fragment length polymor phism (RFLP) analyses (Jeyaprakash et al. 2003). Clones with inserts yielding unique RFLP s were bidirectionally sequenced at the University of Florida Interdisciplinary Core F acility, Gainesville, FL. The resulting sequences were used to design species-specific primers for use in high-fidel ity PCR on individual males in order to determine the frequency of infection with each phylotype Bacterial phylotypes detected in all individual C. thalictri specimens might be considered primary endosymbionts while those found in fewer specimens could be considered secondary endosymbionts or strays from the environment. Primers for each phylotype detected were designed manually (Table 6-1B). Results and Discussion High-fidelity PCR Amplification of Microbial Associates in C. thalictri 16S rRNA was amplified using template DNA isolated from the abdomen, but not the head (including mouthparts) and yielded the expected ~1.5-kb band. This suggests that no symbionts occur in the salivary glands and that the mouthparts were not contaminated with bacteria after surface sterilizati on. No amplification products we re detected using DNA isolated from the head or abdomen of the C. thalictri specimens using primers for Archaea, Fungi including yeast-like organi sms, or Microsporidia. Wolbachia were not detected using either eubacterial 16S rRNA or wspA Wolbachia -specific primers. Given the limited number of C. thalictri samples, false negatives are a possibility but these results suggest the microbial associates of this population are limited to Eubacteria. 169

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Microbial Associates of C. thalictri The eubacterial 16S rRNA PCR products were cloned using 1-L of DNA that had been pooled from nine C. thalictri males. A total of 34 clones were selected at random and analyzed using the RFLP technique. Analysis of Eco R I digests indicated that all clones contained an insert approximately 1.4-kb in length. The Rsa I digests of the 34 clones resulted in five unique banding patterns. Three clones representing each ba nding pattern were sequenced (15 total). Of the 15 sequences, five were unique, indicating that there are at le ast five types of Eubacteria associated with male specimens of the blood-feeding population of C. thalictri occurring in the Primorye Territory of Far Eastern Russia. It is always possible that low frequency phylotypes or specialized Eubacteria will be found in future analyses. One sequence (1457 bp, GenBank accession EF599757) from C. thalictri produced significant alignments to Alcaligenes ( Achromobacter) xylosoxidans (Yabuuchi and Ohyama) strain NFRI-A1of the -proteobacterial family Alcalig enaceae (1485 bp, GenBank accession AB161691.1, Yan et al. 2004). This sequence (EF599757), designated Alcaligenes phylotype 1 from C. thalictri, exhibited a 10.8% sequence divergence relative to the A. xylosoxidans strain NFRI-A1 sequence, which was initially iden tified from the aflatoxin-producing fungus Aspergillus parasiticus Speare (Yan et al. 2004). The sequence from C. thalictri was 99% similar to an uncultured eubacterial speci es (1475 bp, GenBank accession EF509705) from an environmental sample (Flanagan et al unpublished) in the BLASTn results. Alcaligenes ( Achromobacter) xylosoxidans has been identified as part of the culturable microbial community found from the fluid of hooded-pitcher plants, with which some insects form close mutualistic associations (Siragusa et al. 2007). Other Alcaligenes ( Achromobacter) species have been detected in the midguts of malarial mosquito es (Lindh et al. 2005), and associated with 170

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entomopathogenic nematodes (Poinar 1966). Alcaligenes xylosoxidans denitrificans (Ruger and Tan) species have also been used in paratransg enesis experiments, in which these genetically modified gut bacteria were delivered into the gut of the glassy-winged sharpshooter Homalodisca coagulata (Say) in order to interru pt transmission of pathoge ns to crops (Bextine et al. 2004). Given the occurrence of this genus in the guts of other insects, it is possible that this phylotype is associated with the gut of C. thalictri males. A second sequence (1477 bp, GenBank accession EF599759) produced significant alignments to 16S rRNA sequences from -protobacterial species in the family Alcaligenaceae This sequence (EF599759), designated Alcaligenes phylotype 2 from C. thalictri was similar to the homologous portion of the 16S rRNA sequence from Alcaligenes sp. strain IS-67 (9.7% sequence divergence (Table 6-2), 1503 bp, GenBank accession AY346140.1), isolated from an activated sludge system (Zhang et al. 2004). This phylotype from C. thalictri could be a transient from the environment, or a gut associate. A third sequence (1485 bp, GenBank accession EF599758) produced significant alignments with the -proteobacterial family Enterobact eriaceae. This sequence (EF599758), designated Klebsiella phylotype from C. thalictri was similar (0.05% sequence divergence, Table 2) to the homologous por tion of the 16S rRNA gene from K. oxytoca (Flgge) isolate GR6 (1504 bp, GenBank accession AY873801.1), which is ty pically associated with nonleguminous plants (Jha and Kumar unpublished). However, K. oxytoca isolates also are associated with insects, and have been detected in the frass of the leek moth Acrolepiopsis assectella Zeller (Thibout et al. 1995) Both K. pneumoniae (Schroeter) and K. oxytoca have been isolated from pink bollworm Pectinophora gossypiella (Saunders) larvae and adu lts (Kuzina et al. 2002). 171

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Klebsiella species also have been detected in th e Noctuidae (Lighthart 1988) and Pyralidae (Charpentier et al. 1978). It is likely that this phylotype is a gut symbiont of C. thalictri A fourth sequence (1410 bp, GenBank accession EF599760), designated Rhizobium phylotype from C. thalictri, was 99% similar to homologous portions of the 16S rRNA sequence from a Rhizobium species (Heylen et al. 2006) and from an Agrobacterium tumefaciens (Smith and Townsend) sequence (1446 bp, Ge nbank accession DQ468100.1; Wang and Yan unpublished) in the BLASTn results. Subsequent sequence divergence an alyses suggest it is more similar to the Sinorhizobium phylotype 2 (EF599761) from C. thalictri (4.3% sequence divergence, Table 6-2), than it was to the Rhizobium sp. sequence (6.9% sequence divergence, Table 6-2). Species belonging to the Rhizobium-Sinorhizobium group are essential nitrogenfixing bacteria found in legumes (Heckman and Drevon 1987). A fifth sequence (1431 bp, GenB ank accession EF599761) designated Sinorhizobium phylotype from C. thalictri, produced significant alignments to S. morelense of the protobacterial family Rhizobiaceae (1477 bp, GenBank accession AY559079.1). This sequence displayed 6.8% divergence from the S. morelense 16S rRNA sequence (Table 6-2). Bacterial species in the Rhizobium Sinorhizobium group have not been previous ly associated as permanent inhabitants of Lepidopt era, but are found in Tetraponera ants (Kneip et al. 2007). However, recent studies have demonstrated that Rhizobium and Sinorhizobium are closely related to the genus Reichenowia a primary endosymbiont found in blood -feeding leeches (Perkins et al. 2005, Siddall et al. 2004). Siddall et al. (2004) asserted that Reichenowia species might play a role in nitrogen metabolism in the leech, or provide it with other nutrients lacking in a blood meal. It is possible that the Rhizobium and Sinorhizobium phylotypes from C. thalictri represent novel microbial associations similar to those occu rring in blood-feeding leeches; however, the 172

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systematics of these nitrogen-fixing taxa is not resolved and additional sequence data will be necessary to determine their phylogenetic placement. In the diagnostic PCR assay of individual C. thalictri samples using phylotype-specific primers (Table 6-1B), bacter ial sequences representing the Klebsiella and Sinorhizobium phylotypes were detected in 100% of the nine males. Because the Klebsiella phylotype and Sinorhizobium phylotype were detected in all C. thalictri individuals sampled, it is likely that these phylotypes qualify as gut associates. Further study, using methods such as fluorescent in situ hybridization (FISH) are needed to confirm their exact location in C. thalictri Eubacterial sequences representing both Alcaligenes phylotypes and the Rhizobium phylotype were detected in 11%, 33%, and 11% of the nine specimens, respectively. Recent studies have shown the microbiota of the gut in le pidopteran hosts may vary with diet (Broderick et al. 2004), thus, these could be transient contaminants from th e environment or derived from the host plants the moths feed on. The phylotype s found in less than 100% of the individual C. thalictri specimens in this survey are classified as possible, but unconfirmed, gut symbionts based on their similarity to other known gut symbionts in insects. Due to the limited number of specimens available for screening, it is possible that the phylotypes detected in fewer than 100% of the individuals would be more abundant in a larger sample. The screening also could have failed to detect some phylotypes because they occu r in low titer. However, use of a multiple displacement amplification followed by high-fidelity PCR is at least six orders of magnitude more sensitive then standard-allele-specific PCR, allowing us to detect as few as 100 copies of bacterial DNA mixed with insect DNA 100% of the time and as few as 10 copies 50% of the time (Jeyaprakash and Hoy 2004). However, if the in itial primers used were inadequate to detect specific microbial associates, then this survey could have failed to de tect phylotypes. PCR 173

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174 products were not re-amplified due to concerns about contamination. Because negative controls were used in all PCR assays using phylotype -specific primers and no positive bands were observed, as expected, it is unlik ely these sequences were due to laboratory contamination. Wolbachia or any eukaryotic organisms in the specimens screened in our survey were not detected. The failure to find Wolbachia in the C. thalictri specimens is surprising given the prevalence of this bacterium in other butterfly and moth speci es (Tagami and Miura 2004). Some populations of Lepidoptera do consist of both infected a nd uninfected individuals (Tagami and Miura 2004), so it is possible th at screening a larger sample of C. thalcitri specimens could produce different results. It has been speculated that gut endosymbi onts in Lepidoptera larvae are involved in lowering pH, metabolism of toxic plant compounds and pathogen suppression (Broderick et al. 2004). These microbes may also play an important role in pheromone production, mating behavior, longevity, and survival of laboratory-reared colonies in a dults. This initial survey will provide the foundation for future microbial an alyses in both fruit-pi ercing and blood-feeding adult moths.

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Table 6-1. Original and phylotype -specific forward and reverse primers designed to detect microbial sequences in DNA isolated f rom C. thalictri. A Organism (Reference), Forward primer sequence Expected PCR product Annealing Temperature B Phylotype (Genbank accession number) Reverse primer sequence (bp) C A. Primers used in initial survey Archaeabacteria (Arch 21F, Arch 958R) 5TTCCGGTTGATCCYGCCGGA 3 1000 51 (DeLong 1992) 5 YCCGGCGTTGAMTCCAATT 3 Eubacterial 16S rRNA (27F, 1495R) 5 -GAGAGTTTGATCCTGGCTCAG 3 1400 55 (Weisburg et al. 1991) 5 CTACGGCTACCTTGTTACGA 3 Fungal SSU rDNA (NS1, FS2) 5 GTAGTCATATGCTTGTCTC 3 1500 48 (Nikoh and Fukatsu 2000) 5 TAGGNATTCCTCGTTGAAGA 3 Helicosporidia (ms-5, ms-3) 5 GCGGCATGCTTAACACATGCAAGTCG 3 1300 65 (Nedelcu 2001) 5 GCTGACTGGCGATTACTATCGATTCC 3 Wolbachia wspA (81F, 691R) 5 TGGTCCAATAAGTGATGAAGAAAC 3 600 55 (Braig et al. 1998) 5 AAAAATTAAACGCTACTCCA 3 Yeast-like organisms (LS1, LR5) 5 AGTACCCGCTGAACTTAAG 3 2000 50 (Zhang et al. 2003) 5 CCTGAGGGAAACTTCG 3 175 B. Phylotype-specific primers Alcaligenes phylotype (1) 5-GAGAAGA AAAGGTATCCCCTAATACGGGATAC-3 589 65 (EF599759) 5-CTTGCGAGCACTGCCAAATCTCTTCGGC-3 Alacaligenes phylotype (2) 5-GGAAA GAAACGTCGTGGGTTAATACCCCG CGA-3 591 65 (EF599757) 5-CTTGCGAGCACTGCCAAATC TCTTCGGGC-3 Klebsiella phylotype 5-GAAACTGGCAGGCTGGAGTCTTGTAGAG-3 521 65 (EF599758) 5-CAGTCTCC TTTGAGTTCCCGGC CGGACC-3 Rhizobium phylotype 5-GAGACTGGCAGGCTGGAGTCTTGTAGA-3 522 65 (EF599760) 5-CAGTCT CCTTTGAGTTCCCGACCGAATC-3 Sinorhizobium phylotype 5-GTGAAGATAATGACGG TAACCGGAGAAG-3 565 65 (EF599761) 5-CGAACTGAAGGAATACATCTCTGTAATCC-3

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176 Table 6-2. Pairwise sequence dive rgences (uncorrected p) between eubacterial phylotypes from C. thalictri and closely related 16S rRNA sequences using 1410-1504 bp of sequences. 1 2 3 4 5 6 7 8 9 10 1 Alcaligenes sp. IS-67 2 Alcaligenes phylotype 2 .097 3 Alcaligenes xylosoxidans NFRI-A1 .116 .063 4 Alcaligenes phylotype 1 .098 .131 .108 5 Klebsiella oxytoca isolate GR6 .212 .193 .216 .242 6 Klebsiella phylotype .213 .193 .217 .243 .005 7 Rhizobium sp. .229 .229 .200 .224 .242 .242 8 Rhizobium phylotype .246 .225 .243 .258 .241 .242 .069 9 Sinorhizobium morelense .230 .237 .204 .221 .237 .237 .049 .111 10 Sinorhizobium phylotype .250 .234 .248 .260 .233 .232 .109 .043 .068 *Sequences in bold are from C. thalictri.

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CHAPTER 7 COMPARISON OF SHORT-TERM PRESERVATION AND ASSAY METHODS FOR THE MOLECULAR DETECTION OF WOLBACHIA IN THE MEDITERRANEAN FLOUR MOTH EPHESTIA KUEHNIELLA Scientific Note Methods proposed for the preservation of ins ect tissue for DNA analysis have included various concentrations of ethanol, Carnoy s solution, liquid nitrogen, and acetone (Post 1993, Dessauer 1996, Fukatsu 1999, Mtambo 2006). However, little attention has been paid to appropriate storage methods for future detectio n of endosymbiont DNA within an insect host (Fukatsu 1999). Some studies report successful am plification of bacterial DNA in a host after thousands of years (Salo et al 1994, Fricker et al. 1997, Willerslev et al. 2004), but others have reported inconsistent amplification of bacterial DNA due to low titers of the bacteria in the host, difficulties with the DNA extraction process, PCR-i nhibiting substances present in the insect gut, or storage method (Fukatsu 1999, Barnes et al. 2000, Bextine et al. 2004, Hoy and Jeyaprakash, unpublished data). Fukatsu (1999) suggested acet one storage was superior to ethanol as a preservation method for both the amplifica tion of insect host DNA and the DNA of their endosymbionts. Historically, standard PCR has been used to detect Wolbachia and other endosymbionts of arthropods; however, it has been dem onstrated that amplification of Wolbachia DNA can be improved with High-Fidelity (HF) PCR (Jeyapra kash and Hoy 2000). Thus, both the specimen preservation technique and choice of assay me thod could be important in determining the success when attempting to amplify endosymbiotic DNA in an insect host. The goal of this study was to compare molecular methods for the detection of Wolbachia in the Mediterranean flour moth Ephestia kuehniella (Keller) (Lepidoptera: Pyralidae), and potentially other endosymbiotic bacteria in their insect host, in preserved specimens over time. 177

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Materials and Methods Standard, High-Fidelity (HF), and Real-T ime Quantitative (RTQ) PCR methods were used to detect and quantify Wolbachia DNA from E. kuehniella specimens stored under 4 treatment conditions (2 in 95% EtOH and 2 in acetone) over a 2-year storage period. Spectrophotometry readings were taken at each as say (n = 9 over a 2-year period) to ensure consistency of concentration and quality of te mplate DNA for each treatment. Stored samples were compared to fresh specimens at the end of the experiment. A wild-type strain of E. kuehniella was reared on Plodia diet (Silhacek and Miller 1972) in a 16L: 8D photoperiod at 26 C and 70% RH at the USDA Center for Medical, Agricultural, and Veterinary Ento mology, Gainesville, FL. Live E. kuehniella (120 specimens) were anesthetized for a period of 5 min in a -20 C freezer. Once the specimens were immobile, they were placed in 20 mL screw-capped vials (3 per vial). Half of the vials were filled with 95% EtOH, and the remaining vials were filled w ith 100% acetone (Fisher: Atlanta, GA), with a specimen to preservative ratio of 1:20 (based on volume). Half of the vials filled with EtOH were placed in the -80 C freezer and the remaining vials were placed in a rack, covered with a plastic bag to prevent evaporati on, and stored at room temperatur e (25-27C). This procedure was repeated for the vials filled with acetone. After weeks 13, 22, 27, 32, 39, 51, 76, 92, and 101, respectively, 6 specimens from each treatme nt category were removed and placed into clean, disposable Petri dishes and the abdomens we re separated from the thoraces with sterile razor blades. Each abdomen was placed on a Kimw ipe (Fisher: Atlanta, GA) for about 30 sec, and then transferred into a labe led 1.5-mL centrifuge tubes. Abdomens were macerated for 3 min with a grinding pestle made from a 1 mL pi pette tip in a labeled 1.5-mL centrifuge tube. Genomic DNA was extracted from abdomens using a PureGene kit (Gentra Systems: Minneapolis, MN) according to the manufacturers protocol The extracted DNA was re178

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suspended in 250 L of sterile double deionized water. Extractions were stored at -20 C overnight. The quality of the ge nomic DNA for every sample in each of the 4 treatments was assessed using a spectrophotometer w ith a dilution factor of 40 (2 L of extracted DNA per 78 L water). Total DNA concentration and absorbance (260:280 ratio) readings recorded for each set of extractions are listed in Table 71. At the start of the experiment, 20 live E. kuehniella specimens (10 males and 10 females) were tested for the presence of Wolbachia using HF PCR, and all specimens tested positive (100% infection) The HF PCR products were purified using a QIAquick PCR purification column (QIAGEN inc.: Valencia, CA) and were cloned into the plasmid pCR2.1-TOPO according to the manufactu rers protocol (Invitrogen Corporation: Carlsbad, CA). DNA sequencing was performed at the University of Florida ICBR Core Facility using a PERKIN-ELMER Applied Biosystems ABI PRISM Automated DNA sequencer. MacDNASIS software was used to evaluate the sequences (Hitachi Software Engineering America Ltd., San Bruno, California). Wolbachia sequences were identical to Wolbachia sp. group A (Accession #AB024570.1) from E. kuehniella by BLAST. For each specimen in all 4 treatments, we amplified 605 bp of the wsp A gene using wsp-F, 5TGGTCCAATAAGTGATGAAGAAACTAGCTA-3 and wsp-R, 5AAAAATTAAACGCTACTCCAGCTTCTGCAC-3 primers. All PCRs were performed in a total reaction volume of 50 L containing 50 mM Tris (pH 9.2), 16 mM ammonium sulfate, 1.75 mM MgCl2, 350 m dATP, dGTP, dTTP, dCTP, 400 picomo les primers (wsp-F and wsp-R), 1 unit of Pwo and 5 units of Taq DNA polymerases (Jeyaprakash and Hoy 2000). Negative controls were included in all PCRs to test for contamination. The HF PCR cycling profile was: 1 denaturing cycle at 95 C for 2 min, 10 cycles each of denaturation at 94 C for 10 s, annealing at 65 C for 30 s, and extension at 68 C for 1 min and 25 cycles each of denaturation at 94 C for 179

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10 s, annealing at 65 C for 30 s and extension at 68 C for 1 min, plus an additional 20 s added for every consecutive cycle, usi ng a PERKIN-ELMER DNA Cycler 480. A standard PCR analysis also was conducted at each sample date and results were compared to those of the HF PCR analyses (Jeyaprakash and H oy 2000). In order to estimate Wolbachia density in E. kuehniella the wsp A gene copy number was measured from abdomens of 4 individuals from each treatment after a 2-year storage period, a nd compared to the copy number in abdomens from 4 fresh specimens by RTQ PCR using a MyiQ Single-Color Real-time PCR Detection System (Bio-Rad: Hercules, CA). Primers and probes for RTQ PCR analysis of the Wolbachia target DNA (surface protein gene wsp A) were designed using the Primer3 Output v. 0.4.0 software (Rozen and Skaletsky 2000). The forward primer WSP-RTF (5CTATCACTCCATATGTTGGTGTTGGT GTTG-3) corresponded to the region from base 327356 of the wsp A sequence and the revers e primer WSP-RTR (5CTCCTTTGTCTTTCTCACCAACGC TTTTAT-3) to the region from 519-548 of the same sequence. The length of the amplification product was 222 bp. The Taqman real-time PCR protocol was performed in a final volume of 25 L and contained: 1 L of template DNA, 10 L of Power SYBR Green PCR Master Mix (Appl ied Biosystems: Foster City, CA), 7.24 L double deionized water, 0.16 L Accuzyme DNA polymerase (Bioline: Randolph, MA), and 0.8 L of each primer, and was carried out in 96-well plat es (Applied Biosystems: Foster City, CA) that had been sealed with film, and centrifuged for approximately 30 sec (100 rpm) prior to RTQ PCR. The RTQ PCR cycling profile consisted of 10 min at 95C followed by 50 cycles of 10 s at 95C, 30 s at 60C, 30 s at 68C, followed by a melt cycle for which conditions were 15 s at 95C, 1 min at 65C, and 15 s at 95C. A sta ndard curve was calculat ed by using a standard plasmid sample that contained the wsp A gene at concentrations of [10 2 to 10 9 ] copies/ l. The 180

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number of molecules in the samples was determined from the threshold cycles ( C T ) in the PCR based on the standard curve. Negative contro ls were included in all PCRs to test for contamination. Results Wolbachia ( wsp A) was detected in all E kuehniella individuals (n = 6) in each treatment (n = 4) over the 2-year period (9 total sample times) using both standard and HF PCR (data not shown). The average wspA copy number (Table 7-1) found in E kuehniella abdomens (n = 20) for each of the 4 treatments and from 4 abdomens from fresh specimens using RTQ PCR was lower than previously reported in the testis of different male strains of E. kuehniella ( groEL copy number in testis: 5.2 7.5 x 10 6 2.4; Ikeda et al. 2003). This is not su rprising given the potential variation in abundance of Wolbachia within an insect host de pending on strain, sex, life cycle, and tissues or organs sa mpled (Ijichi et al. 2002). The wsp A copy number in each of the 20 individuals sampled was not si gnificantly different among pres ervation treatments based on C T values from the RTQ PCR (ANOVA, F= 0.53, df= 4, P= 0.72). The HF PCR amplicon band intensity for th e material stored in 95% EtOH at room temperature for 2 years appeared lower for 3 out of the 6 specimens sampled (compare figures 71A and 7-1B). When compared to HF PCR, a reduction in band intensity for standard PCR after the 2-year storage period for all treatmen ts was also observed (data not shown). The average DNA concentration/sample taken at the last sample date (week 101) ranged from 17 to 23 g/mL and the 260:280 ratio of the stored samples ranged from 0.8 to 1.2; fresh specimens had a DNA concentration of 25 and a 260:280 ratio of 1.7 using the same extraction protocol (Table 7-1). Averag e total DNA concentrations and 260:280 ratios taken at the last sample date (week 101) were not significantl y different between treatments (ANOVA, F= 1.4, df= 4, P= 0.28 and ANOVA, F= 1.7, df= 4, P= 0.18, respectively). 181

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Surprisingly, over the 2-year storage period, a regression analysis showed an increase in total DNA concentration over the sample dates (data not shown) (slope = 0.340, y-intercept = 8.360, and R 2 = 0.05 for specimens stored in acetone at RT; slope = 1.365, y-intercept = 7.203, and R 2 = 0.5 for acetone at -80C; slope = 1.469, y-intercept = 5.750, and R 2 = 0.3 for 95% EtOH at RT; slope = 1.132, y-intercept = 8.226, and R 2 = 0.3 for 95% EtOH at -80C), perhaps due to dehydration of the specimens over time allowing for easier maceration of spec imens. A decrease in the quality of the total DNA was observed (dat a not shown) for all 4 treatments (slope = 0.005, y-intercept = 1.28, and R 2 = 0.005 for specimens stored in acetone at RT; slope = -0.071, y-intercept = 1.62, and R 2 = 0.5 for acetone at -80C; slope = -0.021, y-intercept = 1.55, and R 2 = 0.1 for 95% EtOH at RT; slope = -0.070, y-intercept = 1.55, and R 2 = 0.3 for 95% EtOH at -80C) suggesting the quality of the DNA was compromise d over time due to storage in each of the 4 treatments. Discussion These results are consistent with previous studies (Fukatsu 1999) suggesting acetone and 95% EtOH are adequate short-term specime n preservation methods for detection of endosymbiotic bacterial DNA in an insect host. Although not statistic ally significant, an apparent reduction in Wolbachia wsp A copy number and HF PCR band intensity of the symbiont for specimens stored in 95% Et OH at room temperature was obser ved (Fig. 7-1A, Table 7-1). Averages in the total DNA concentration and purity of the stored specimens at week 101 were not significantly different among replicates from the same treatment nor were they significantly different between treatments. Regression an alyses revealed an increase in total DNA concentration over time for each treatment, and a decrease in absorbance over time. Additionally, a reduction in band intensity was observed for specimens stored in 95% EtOH at room temperature at the last sample date (week 101), suggesting long-term storage in 95% EtOH at 182

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183 room temperature may not be an optimal storage method as it may further damage the DNA or inhibit the PCR. The results reported here are specific to E. kueniella but they indicate that when Wolbachia infection density in the host is high and the DNA extraction method is consistent, both standard and HF PCR are adequate for detection of Wolbachia after a 2-year storage period in both acetone and 95% EtOH.

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Table 7-1. Summary of RTQ PCR a nd spectrophotometry data for E. kuehniella specimens stored for a 2-yr. period compared to fresh specimens. Storage method wspA copy number in abdomen Total DNA concentration (g/mL) 260:280 (mean SD) (mean SD) 1 (mean SD) 1 Acetone room temperature 4.422 x 10 5 1.276 x 10 4 17 4.0 1.2 1.0 Acetone -80C 4.361 x 10 5 3.973 x 10 4 20 1.0 1.2 0.7 95% EtOH room temperature 3.441 x 10 5 2.295 x 10 5 18 5.5 1.2 0.4 95% EtOH -80C 4.245 x 10 5 6.913 x 10 4 23 7.3 0.8 0.2 Fresh specimens 2 4.202 x 10 5 2.218 x 10 4 25 9.0 1.7 0.0 Acetone combined 3 4.391 x 105 2.751 x 104 19 3.2 1.2 0.8 95% EtOH combined 3.843 x 10 5 1.627 x 10 5 21 6.5 1.0 0.4 Room temperature combined 3.931 x 10 5 1.592 x 10 5 23 8.3 1.5 0.2 -80C combined 4.303 x 10 5 5.256 x 10 4 22 5.1 1.0 0.5 *Total DNA concentration and 260:280 values are averages of individuals sampled (n = 6) and assayed over the 2-year period (n = 9 datapoints). Fresh Specimens (n = 5) were analyzed at week 101. Combined values indicate aver ages of treatments combined (e.g., stored in acetone at room temperatur e + stored in acetone in -80C freezer). 184

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RT -80C 0 _____________ ____________ 0 IV 3 4 5 6 7 8 9 10 11 12 13 14 A B Figure 7-1. Examination of DNA preserva tion and amplification by HF PCR of the wsp A gene fragment (605 bp) in E. kuehniella (A) HF-PCR amplifi cation of 12 individual E. kuehniella specimens following a 2-yr stor age period in 95% EtOH at room temperature (lanes 3 8) and at -80C (l anes 9 14). Blank lanes = 0, negative control lanes = -. (B) HF PCR amplification of 12 individual E. kuehniella specimens following a 2-yr storage period in acetone at room temperature (lanes 3 8) and at 80C (lanes 9 -14). IV = HyperLadder (Bioline MA), blank lanes = 0, negative control lanes = -. 185

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CHAPTER 8 PERSPECTIVES This chapter describes important experiences during my time as a Ph.D. student in the Entomology and Nematology Department at the Univer sity of Florida. This section is meant to be a self-evaluation and discussion of what I l earned, challenges I faced, and how I might handle similar challenges in the future. I arrived in Florida in the summer of August 2004 having just completed my Masters Degree in Dr. Susan Wellers lab in the Entomology Department at the University of Minnesota. At the time, my background was in system atics, morphology, taxonomy, and biogeography and my goals were to augment these skills by learni ng techniques in molecular biology. My goals were to reconstruct a phylogeny of fruit-piercing and blood-feedi ng moths to test the hypothesis of a directional progression from nectar feeding to blood feeding in the group known as Calpini. When I came to UF I had a large loan of specime ns of calpine moths fr om the National Museum of Natural History (Smithsonian) so I could begi n the project immediately. I began by dissecting relevant taxa, coding morphological characters, and had a preliminary phylogeny within the first year. At this time, Dr. Branham encouraged me to also survey the characters of the proboscis using the optics system in his laboratory and also SEM equipment at DPI. This was incredibly challenging, but through trial and error and much patience I finally learned how to use both types of equipment. I was extremely fortunate to have Dr. Branhams training in how to use the digital equipment in his laboratory for my dissertation. The proboscis images taken and descriptions of the structures observed has now turned into a size able chapter in my dissertation and resulted in an important publication with Dr. Branham and an international collaborator, Dr. Hans Bnziger. While most of my coursework was completed during my Masters program, as part of my Ph.D. at UF I completed the following course s: Advanced Invertebrate Zoology, Medical and 186

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Veterinary Entomology, Technique s in Insect Systematics, Ins ect Molecular Biology, Molecular Systematics, Phylogenetic Met hods, Population Genetics Seminar, Insect Symbiosis Seminar, and Protein Chemistry and Molecular Cloning Summer Laboratory course. These courses increased my knowledge in entomology, systematics, and molecular biology. The Insect Molecular Biology course taught by Dr. Hoy was of great interest to me as I gained further exposure to microbe-insect interac tions through topics covered in class discussions. I found this subject very interesting and im mediately decided to discuss th e possibility of conducting an endosymbiont survey on fruit-piercing and blood -feeding moths. Dr. Hoy encouraged me to pursue this and offered me bench space, training, and supplies in her laboratory. I began working on this project right away, however, I did not have fresh specimens of blood-feeding moths at the time and so I used freshly collected material from a previous expedition to Nepal. Initially, this work was very stre ssful as I had little to no previ ous at the bench as was trying to re-learn molecular biology at fast pace. Additio nally, the specimens I was using for the survey of microorganisms were testing negative and it wa s unclear whether these results were true or false positives. We were unsure whether the moths had been stor ed properly for extraction of endosymbiont DNA. During this time I devel oped a good working relationship with Dr. Hoy where we had numerous discussi ons about how we could set up an experiment to answer the question of which method of specimen storage is be st for the extraction of microbial DNA. This experiment would later become a technical chapter of my dissert ation and resulted in another publication. This chapter taught me a lot about the scientific methods and problem solving. During this study I learned how to conduct both Hi gh Fidelity (HF) and real time quantitative (RTQ) PCR. 187

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During the spring of 2006 I was fortunate to receive a grant from the Explorers club to travel to Far Eastern Russia to look study adu lt feeding behavior in three species from the vampire moth genus Calyptra This was a very successful fiel d trip in that I learned which species in this region actually feed on blood and which do not. I was also able to acquire specimens for both my phylogenetic study and for th e endosymbiont survey. Thus, while I was working on the technical chapter I began working on an endosymbiont survey of a fruit-piercing and blood-feeding moth, Calyptra thalictri. Through this study I learne d several important skills in molecular biology including clon ing and RFLP analys is. This study became another chapter of my dissertation and has resulte d in another publication. I was fortunate to be supported by Dr. Hoy for both molecular biology chapters and most importantly, was trained in bench skills that most systematic students are not exposed during their Ph.D.s. My experiences in Dr. Hoys lab were extremely positive in that I also had the opp ortunity to learn from her former student, Jason Meyer, and also teach other graduate students and postdoctoral researchers methods I learned while working in her laboratory. During my Ph.D. I had the opportunity to TA the course Biology of Lepidoptera where I designed and taught the laborat ories and gave a lecture on insect heads and mouthpart morphology. I also volunteered as a teaching as sistant for Dr. Jim Lllyods Natural History honors course for two semesters. I plan to co-i nstruct a course in insect biology and possibly a summer field course or seminar during the seco nd year of my postdoctoral position at the University of Minnesota. During my Ph.D., I always had financial s upport from both Dr. Bra nham and Dr. Hoy for which I am incredibly thankful. Despite the av ailability of funds thr ough Drs. Branham and Hoy and the department, I always felt it was important to try and raise my own funds to support my 188

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projects. While a Ph.D. student at UF I submitte d several small and large grant proposals to try and support my salary, laboratory work, and fieldw ork. I was successful in raising funds from several granting agencies includ ing the National Park Service, the National Science Foundation, and the National Geographic Society. I also raised $3,000 from the Systematics Research Fund to pay for a trip to the British Museum of Natu ral History. I submitted two proposals to the EPA to try and cover my student stipend, but unfor tunately was unsuccessful. I also had the opportunity to write and submit a grant proposal to the DEB panel at the National Science Foundation with Dr. Susan Weller. This experience was incredibly useful as I now feel prepared for future grant writing in an academic position. Through my grant writing experiences I le arned several valuable skills including professional communication. This is incredib ly important because many funding agencies require supporting documentation before reviewing and funding proposals. I have been fortunate to have the support of all my committee memb ers and other members of the scientific community in this area and I am certain that their agreeing to write good letters of support was instrumental in my receiving funding over the y ears. My committee has also taught me the importance of writing and publishing the results of my research as the data comes in and not to wait until the end to write everything as the publi cation process is tedious and time-consuming. I think this is one of the most important philosophies a committee can pass down to the students they advise because it prepares students for the real world and teaches us how to be efficient with our time. This is important given academics is a competitive environment that requires individuals to be successful in many areas at the same time while continuing to raise grant money and publish the results in peer-reviewed journals. My committee has supported me from the very beginning of my Ph.D. and given me solid scholarly advice on all occasions. They have 189

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and trained me and prepared me for a future in an academic position. At times all graduate students can become overwhelmed by the tasks at hand and question whether or not they are going to make it. My committee members have helped me overcome these fears and gain the confidence needed to be a successful scientist. I am very pleased that my dissertation chapters covered the aspects I set out to cover in the beginning of my P h.D. program and much more. My time in the Entomology and Nematology Department was a positive one. I am happy with the research topic that I chose for my dissertation and that I was able to publish results as the projects were completed. I must extend my greatest appreciation to all members of my committee for both their personal and professional support; I consider them my academic family and friends that I hope to carry with me for the remainder of my career. My long-term career goals include a position at a major university teaching courses in Entomology and conducting research on economically important moths, especially fruit-piercing and blood-feeding moths. I intend to apply syst ematics to other areas of entomology including Integrated Pest Management (IPM), Risk Asse ssment, Biological Control, and Conservation Biology. This work will also apply to othe r areas of Entomology such as Medical and Veterinary Entomology, by inve stigating disease vector poten tial in skin-piercing and bloodfeeding moth species. My course of study at the University of Florida has allowed me to gain important insight into the evol ution of an environmental, eco nomic, and agricultural model organism as well as link many fields of entomology. 190

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191 APPENDIX A DATA MATRIX USED TO PRODUCED TREES BASED ON MORPHOLOGICAL DATA

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Taxa Character A. flava 120010001000?0021100????????????????????????0B090?0011110???0?10?? A. mesogona 120010001000?00211000??B0?1001190280391010000B090?0011110???0?1122 S. libatrix 120030101000?202010011?50?000116021014630000040?0?010?220???0?10?? H. hormos 010011000050?202210013?60?A00111009559430000080A0?010?0?0???0?0100 H. monilis 010011000050?20221000??30?B0010?009559430000181?110113110???0?10?? He. hieroglyphica 12012?002000?00111010??41270010?11455040300001140?000?0?0???0?10?? He. sittica 12012?002000?2?111010??41370010?314550400000?????????????????????? Phy. callitrichoides 010011000020?20134002?290?3001110??061100020083?1201100?0???0?0111 C. albivirgata 12010?1100010200102115?60?300110012610330200050?16010?0?0???0?00?? C. bicolor 12010?1100010200102115?20?3001100126103300100?0?11000?0?0???0?00?? C. canadensis 12010?1100010220201114?20?30011200?6153202000A1110010?0?0???0?10?? C. eustrigata 12010?1100010101120115?60?300110012610330210300?0?010?0?11700?00?? C. fasciata 12010?1100010200100115?20?3001100???10330110300?13010?0?0???0?00?? C. fletcheri 12010?1100010200102115?60?300110012610330100?????????????????????? C. gruesa 12010?1100010200122115?20?3001100126103302000A0?10000?0?11200?00?? C. hokkaida 12010?110001000112213?020?3001100???103302100A1?1600110?0???0?00?? C. lata 12010?1100010200122115?20?300110012610300010341?16010?0?0???0?00?? C. minuticornis 12010?1100010001122110?60?300110012610330200300?0?010?0?0???0?00?? C. ophideroides 12010?1100010201122115?20?3001100???10300000300?16010?0?0???0?00?? C. orthograpta 12010?1100010002310115?60?300110012610010000300?17010?0?0???0?10?? C. parva 12010?1100010001122149?20?300110012610330200304?0?00140?0???0?00?? C. pseudobicolor 12010?110001020010211A?20?30011000?610000010300?18010?0?0???0?00?? C. subnubila 12010?1100010200102115?20?3001100??????30210300?19000?0?11600?00?? C. thalictri 12010?1100010200322118?60?300110006610310200000?0?000?0?0???0?00?? E. anguina 12032?1101311?025101????????????????????????001?0?010?0?0???0?10?? E. aurantia 12032?110131100251200??30?4031140120555001000E0?0?110?0?0???0?10?? E. boseae 12032?1101311?0251010??30?602118015452500010091?15030?0?0???0?00?? E. cocalus 12032?110131100251010??30?90111401205511000007060?100?0?0???0?10?? E. dividens 12032?110131100251010??20?30111401245550011000070?110?0?0???0?10?? E. fullonia 12032?110131100251010??A0?40111801705700000006100?100?0?0???0?10?? E. homanea 12032?110131100251010??310302118012?555100000F0?0?000?0?0???0?10?? E. jordani 12032?110131100251010??60?30211401245551001006100?100?0?0???0?10?? E. materna 12032?1101311002510017?30?302114012055500010000005100?0?11800?10?? E. procus 12032?11013110025101????????????????????????00030?000?0?0???0?10?? E. salaminia 12032?110131100251200??21030211401205550001000050?110?0?0???0?00?? 192

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193 Taxa Character E. tyrannus 12032?110131100251110??30?602114012455520100000814000?0?0???0?10?? G. correcta 12010?1100110000132112?20?200111215111020001022?1300150?01090?00?? G. incurva 12010?1100110020132112?20?200111215105020011122?1000150?0???0?00?? G. indentata 12010?1100110000102112?20?300111015108000011020?0?400?0?0???0?00?? G. mexicana 12010?1100110000132112?60?300110010?10000011022?0?00150?10210?00?? G. nutrix 12010?11001100?0132112?70?200110215100000010020?0?020?0?0???0?00?? G. parens 12010?1100110000132112?20?300110215118000011022?1001150?0???0?00?? G. sicheas 12010?1100110000132012?20?200111215118000001020?0?610?0?00210?00?? G. sinaldus 12010?1100110000131112?70?400110015100000211020?10510?0?0???0?00?? G. unica 12010?1100110000132112?20?400110215108000211022?1001150?0???0?00?? G. uxor 12010?1100110000132112?20?200111215118?????1020?13000?0?0???0?00?? Gr. regina 12?32??????1000251000??111300117003345040010211?0?700?0?0???0?00?? O. argyrosigna 12010?111001000040211B?80?801110012123300000080?0?800?0?11400?00?? O. emarginata 12010?111001001040210??80?801110012123300000080?0?020?0?11110?00?? O. excavata 12010?1110010100402112?80?800110012125300000080?12010?0?11410?00?? O. excitans 12010?1110010002311112?80?300110012123300010080?12010?0?11010?00?? O. glaucohelia 12010?11100100?2312112?80?400110012125340000080?12810?0?11500?00?? O. nobilis 12010?1110010000401112?80?800110012125300000080?0?000?0?11410?00?? O. provocans 12010?1110010?0?4?11????????????????????????080?0?000?0?11610?00?? O. rectristria 12010?111001010040212?180?100110012123300000083?0?02100?11310?00?? O. serpans 12010?11100100?142210??810100110012125310010?????????????????????? O. striolata 12010?111001000040011B?80?400110012120300010080?0?010?0?11010?00?? O. triobliqua 12010?11100101?040210??80?810110012525300000080?0?02100?11210?00?? P. casta 12110?1100210002410110?20?100111005250101110021?1100120?0???0?00?? P. coelonota 12110?1100210002411117?20?300115015?51100000030?0?200?0?0???1110?? P. compressipalpus 12110?1100210222410116?00?5101110??2561300000C1B10010?0?0???1310?? P. dimorpha 12110?1100210002412117?20?500115005050100000030?0?90120?0???0?00?? P. incitans 12110?1100210??2412117?20?300111005201100000080?0?330?0?0???0?00?? P. miranda 12110?110021000241101??20?300111005201120000431210210?0?0???1300?? P. repellens 12110?1100210002412117?20?5001???050???00000030?0?90120?0???0?00??

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194 APPENDIX B DATA MATRIX USED TO PRODUCE TREES BASED ON MORPHOLOGICAL AND MOLECULAR DATA

PAGE 195

Aflava 120010001000?0021100????????????????????????0B 090?0011110?? ?0?10?? Amesogona 120010001000?00211000??B0?1001190280391010000B090?0011110???0?1122 Slibatrix 120030101000?202010011?50?000116021014630000040?0?010?220???0?10?? Hhormos 010011000050?202210013?60?K00111009559430000080K0?010?0?0???0?0100 Hmonilis 010011000050 ?20221000??30?B0010?00955943 0000181?110113110???0?10?? Hsittica 12012? 002000?2?111010??41370010?314550400000?????????????????????? Phycallitrichoides 010011000020?20134002? 290?3001110??0 61100020083?1201100 ?0???0?0111 Calbivirgata 12010?1100010200102115?60?300110012610330200050?16010?0?0???0?00?? Cbicolor 12010?1100010200102115?20?3001100126103300100?0?11000?0?0???0?00?? Ccanadensis 12010?1100010220201114?20?30011200?6153202000A1110010?0?0???0?10?? Gsinaldus 12010?1100110000131112?70?400110015100000211020?10510?0?0???0?00?? Gparens 12010?1100110000132112?20?300110215118000011022?1001150?0???0?00?? Gincurva 12010?1100110020132112?20?200111215105020011122?1000150?0???0?00?? Gcorrecta 12010?1100110000132112?20?200111215111020001022?1300150?01090?00?? Clata 12010?1100010200122115?20?300110012610300010341?16010?0?0???0?00?? Cthalictri 12010?1100010200322118?60?300110006610310200000?0?000?0?0???0?00?? Eaurantia 12032?110131100251200??30?4031140120555001000E0?0?110?0?0???0?10?? Ejordani 12032?110131100251010??60?30211401245551001006100?100?0?0???0?10?? 195 Ematerna 12032?1101311002510017?30?302114012055500010000005100?0?11800?10?? Esalaminia 12032?110131100251200??21030211401205550001000050?110?0?0???0?00?? Etyrannus 12032?110131100251110??30?602114012455520100000814000?0?0???0?10?? Gindentata 12010?1100110000102112?20?300111015108000011020?0?400?0?0???0?00?? Gnutrix 12010?11001100?0132112?70?200110215100000010020?0?020?0?0???0?00?? Gsicheas 12010?1100110000132012?20?200111215118000001020?0?610?0?00210?00?? Guxor 12010? 1100110000132112?20?2001 11215118?????1020?13000? 0?0???0?00?? Oemarginata 12010?111001001040210??80?801110012123300000080?0?020?0?11110?00?? Oexcitans 12010?1110010002311112?80?300110012123300010080?12010?0?11010?00?? Onobilis 12010?1110 010000401112?80?800110012125 300000080?0?000?0 ?11410?00?? Orectristria 12010?111001010040212?180?100110012123300010083?0?02100?11310?00?? Oserpans 12010?11100100?142210??810100110012125310010?????????????????????? Pcasta 12110?1100210002410110?20?100111005250101110021?1100120?0???0?00?? Pcoelonota 12110?1100210002411117?20?300115015?51100000030?0?200?0?0???1110?? Pcompressipalpus 12110?1100210222410116?00?5101110??2561300000L1B10010?0?0???1310?? Pdimorpha 12110?1100210002412117?20?500115005050100000030?0?90120?0???0?00??

PAGE 196

[COI] Ematerna AACATTATATTTTATTTTTG GTATTTGAGCCGGTATAGTAG GAACTTCTCTTAGTTTATTA ATTCGAGCTGAATTAGGAA ATCCAGGATCATTAATTGGAGATGATCAAATTTATAATACTATTGTTACAGCTCATGCTTT CATTATAATTTTTTTTATA GTAATACCTATTATAATTGGAGGATTCGGAAATTGATTA ATTCCTCTTATATTAGGTG CTCCTGATATAGCTTTTCCTCG TATAAATAATATAAGTTTTTGACTTCTT CCCCCTTCTTTAACTCTTCTTATTTCAAGAAGAATTGTAGAAAATGGAGCAG GAACAGGATGAACAGTATATC CCCCACTATCATCTAATATTGCACATAG AGGTAGATCTGTAGATTTAGCTATTTTTTCT TTACATTTAGCTGGTATTTCATCAATTTTAGGAGCAATTAATTTTA TTACTACAATTATTAAT ATACGATTAAATAATTT ATCATTTGATCAAATACCCCTATTTGTTT GAGCTGTTGGAATTACTGCATTTTTA TTACTTCTTTCTTTACCTGTTCTAGC AGGAGCTATTACTATACTTTTAACAGATCGAAATTTAAATA CATCATTTTTTGATCCTGCTGGTGGAGGAGATCCCATTC TTTATCAACATTTATTT??????? Aflava AACATTATATTTTATTTTTG GTATTTGAGCAGGAATAGTCGGAACTTCAT TAAGTATATTAATTCGAGCAGAATTAGGTA ATCCAGGATCTTTAATTGGTGACGAT CAAATTTATAATACTATTG TTACTGCCCATGCTTTT ATTATAATTTTTTTCATAG TTATACCTATTATAATTGGTGGATTT GGAAACTGATTAGTACCTCTTATATTA GGAGCTCCAGATATAGCTTTTCCTCGA ATAAATAATATAAGATTTTGACTTCTTCCCCCTTCTTTAAT TCTTTTAACTTCAAGAAGAAT TGTAGAAAATGGAGCAGG AACAGGATGAACAGTTTATCC CCCACTTTCATCTAATATTGCTCATAGAGGAAG CTCAGTAGATCTTGCTATTTTTTCCC TTCATTTAGCAGGTATTTCATCAATTTTAGGAGCTATTA ATTTTATTACAACAATCATTAATATACGATTAAATAATTTA TCATTTGATCAAATACCTTTA TTTGTTTGAGCAGTAGGAATCACAGCATTTTTATTATTATTA TCACTACCAGTATTAGC AGGAGCTATTACAATATTATTAACAGAT CGTAATTTAAATACTTCTTTTTTTGAT CCTGCAGGAGGTGGAGATCCTATtC TTTATCAACACTTATTT??????? 196 Amesogona ???????????????TTTTGGTATTTGAGCAGGT ATAGTTGGAACTTCATTAAGTATATTA ATTCGAGCAGAA TTAGGAAACCC AGGATCTTTAATTGGAGATGATCAAATTTATAATACTATTGT TACTGCTCATGCTTTTATT ATAATTTTTTTTATAGTTAT ACCTATTATAATTGGAGGATTCGGAAATTGATTAGTACCA CTTATATTAGGAGCACCTGATATAGCTTTCCCCCGAATA AATAATATAAGATTCTGACTTCTTCCACCTTCACTTATTTT ATTAACTTCAAGAAGAATTGTAGAAAATGGAGCAGGAA CAGGATGAACAGTTTATC CCCCACTTTCATCTAAT ATTGCTCATAGAGGAAGCTCA GTAGATTTAGCAATTTTTTCACTA CATTTAGCAGGAATTTCATCCATTTTAGGAGCTATTAATTTTATTACTACAAT TATTAATATACGATTAAATAATCTATC ATTCGATCAAATACCATTATTTGTTTGA GCTGTTGGTATTACAGCA TTTTTATTATTATTATCTCTTCCTGTATTAGCTGG AGCAATTACTATATTATTAACTGACCG TAATTTAAATAC???????????????? ?????????????????????????????????????????????? Calbivirgata ????????????????????????????????? ????????????TTCTCTAAGCTTATTAATTCG AGCTGAATTAGGTAATCCTGGATCTTTAA TTGGAGATGATCAAATTTATA ATACTATTGTAACAGCTCACGCTTTTATT ATAATTTTTTTTATAGTTATACCTATTATAA

PAGE 197

TTGGAGGATTTGGAAATTGAC TTGTACCTTTAATATTAGGAGCTCCTGAT ATAGCTTTCCCTCGAATAAATAATATAAGT TTTTGACTTCTTCCCCCTTCTTTAACTC TTCTAATTTCTAGAAGAATTGTA??????????? ??????????????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ???????????????????????????? ????????????????????????? ????????????????? Cbicolor AACATTATATTTTATTTTTG GAATTTGAGCAGGAATAGTT GGAACCTCATTAAGACTATTA ATTCGAGCTGAATTAGGTA ATCCTGGATCTTTAATTGGAGATGATC AAATTTATAATACTATTG TAACAGCTCATGCTTT CATTATAATTTTTTTTATAG TTATACCTATTATAATTGGAGGATTTGG AAATTGACTTGTCCCTCTTATATTAGGAGCACCAGATATAGCTTTCCCTCGA ATAAATAATATAAGTTTTTGACTTCTTCCCCCCTCTTTAACTCTTCTA ATTTCTAGAAGAATTGTAGAAAATGGAGCAGG AACAGGATGAACAGTATA CCCCCCCCTTTCATCTAATATTGCACATA GAGGAAGATCAGTA GATTTAGCTATTTTTTCA TTACATTTAGCAGGAATTTCA TCAATTTTAGGAGCAATT AATTTTATTACAACAATTATTA ATATACGATTAAATAATTT ATCATTTGACCAAATACCTTT ATTTATTTGAGCAGTAGGAATTA CAGCATTCTTATTATTATTATCTTTACCTGTTTTAGC TGGAGCTATTACAATACTTTTAACAGATCGAAATTTAAATA CATCTTTCTTCGATCCTGCT GGAGGAGGAGAtCcTATTCT TTACCAACATTTATTT??????? Clata AACATTATATTTTATTTTTG GAATTTGATCAGGAATAGTTGGAACTTCATTAAGATTATTA ATTCGAGCTGAATTAGGTA ATCCAGGATCTTTAATTGGAGATGATCAAATTTATAACACTATTG TAACAGCTCATGCTTTT ATTATAATTTTTTTTATA GTTATACCTATTATAATTGGAGGATTTGGAAATTGACTTGT ACCCCTCATATTAGGAGCCCC TGATATAGCTTTCCCCCG AATAAATAATATAAGTTTTTGACTAC TTCCCCCCTCATTAACCCTTTTAATTTCTAGAAGAAT TGTAGAAAATGGAGCAG GAACAGGATGAACAGTGTA TCCCCCCCTTTCATCT AATATTGCACATAGAGGAAGT TCTGTAGATTTAGCTATTTTTTCC CTTCATCTAGCAGGAATTTCATCAATTTTAGGAGCAATTAATTTTATTACAACAATTATTAAC ATACGATTAAATAATTT ATCATTTGATCAAATACCTTTATTTATTT GAGCAGTAGGAATTACAGC ATTCTTACTTTTATTAT CTTTACCTGTTTTAGC TGGAGCTATTACTATACTTTTAACAGATCGAAATTTAAATA CATCTTTTTTTGATCCTGCT GGAGGAGGAGATCcTATTC TTTATCAACATCTATTT??????? 197 Cthalictri AACATTATATTTTATTTTTGGAATTTGAGCAGGAATAGT TGGAACTTCACTAAGATTA TTAATTCGAGCCGAACTAGGA AATCCAGGATCTTTAATTGGAGATGATCAAATTTATAATAC TATTGTAACAGCTCATGCTTTCATTATAATTTTTTTTAT AGTTATACCTATTATAATTGGAGGATTT GGAAATTGATTAGTACCCCTTATATTAGGAGCTCCTGATATAGCTTTCCCTC GAATAAATAATATAAGTTTTTGACTCCTCCCCCCTTCTTTAAC TCTTCTAATTTCCAGAAGAA TTGTAGAAAACGGAGCA GGAACTGGATGAACAGTATATCCCCCCCTTTCATCAAATATTG CACATAGAGGAAGTTCTGTAGATTTAGCTATTTTTTC ATTACATTTAGCAGGAATTTCATCAATTTTAGGAGCAATTAATTTTATTACAACA ATTATTAATATACGACTAAATAATT TATCATTTGATCAAATACCTTTATTTAT TTGAGCAGTAGGAATTACAGCATTTCTA CTCTTATTATCTTTACCTGTTTTAG

PAGE 198

CTGGAGCTATTACTATACTTT TAACAGATCGAAATTTAAA TACATCTTTTTTTGATCCTGCT GGTGGAGGAGATCCTATT TTATATCAACATTTATTT??????? Eaurantia ??????????TTTATTTTTGGTATTTGAGCAGGtAtAGTAGGAACTTCACTCAGTTTATTAATTCGAGCTGAATTAGGAAATCC AGGATCATTAATTGGAGATGACCAAATTT ATAATACTATTGTTACAGCTCATG CTTTCATTATAA TTTTTTTTATAGTAA TACCTATTATAATTGGAGGATTTGG AAATTGACTAGTACCTCTTATATTAGG AGCTCCTGATATAGCTTTCCCTCGAATA AATAATATAAGTTTTTGACTTCTCCCCCC TTCTTTAACTTTATTAATTTCAAGAA GAGTTGTAGAAAATGGAGCAGGAAC TGGATGAACAGTTTACCCCCCACTAT CATCTAATATTGCACATAGAGGAAGT TCAGTAGATTTAGCTATTTTTTCATTAC ATTTAGCTGGTATTTCATCAATTTTAGGAGCTATTAATTTTATTACAACAATTATT AACATACGATTAAATAACTTATCA TTTGATCAAATACCATTATTTATTTGAGCTGTAGGAATTACA GCATTCTTATTATTATTATCTTTACCAGTTTTAGCAGGT GCTATTACCATACTTTTAACAGACCG AAATTTAAATAC?????????????????????????????????????????????????????????????? Ejordani ?????????????ATTTTTGGTATTTGAGCAGGtA tAGTAGGAACTTCACTT AGTTTATTAATTCGAGC TGAATTAGGAAATCCA GGATCACTTATTGGAGATGATCAAATTTATAATACTATTGTT ACAGCTCATGCTTTTATTATAATTTTTTTTATAGTAATA CCTATTATAATTGGAGGATTTGGAAAT TGATTAGTACCTCTTATATTAGGAGCCCCTGATATAGCTTTCCCCCGAATAAA TAATATAAGTTTTTGACTT CTTCCCCCTTCTTTAACTCTTT TAATTTCAAGAAGAATTGTAGAAAACGGAGCAGGAACTG GATGAACAGTCTATCCCCCAC TTTCATCTAATATTGCTCATAGAGGAAGTTCAGTAGATTTA GCTATTTTTTCACTACAT TTAGCTGGTATTTCATCAATTTTAGGAGCCATTAATTTTATT ACAACAATTATTAATATACGATTAAATAATTTATCATT CGATCAAATACCACTATTTATTTGAGCT GTTGGAATTACTGCATTCTTATTACTTCTTTCTTTA CCTGTCTTAGCAGGTGC TATTACTATACTTTTAACAGAT CGAAATTTAAATAC???????????????? ??????????????????????????? ??????????????????? 198 Esalaminia ???????????????????????TTTGAGCAGGTATAGTA GGAACTTCTCTTAGTTTATTAATTC GAGCTGAATTAGGAAACCCCGG AtCATTAATTGGAGAtGATCAAATTTATAATACTATTGTTA CAGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCT ATTATAATTGGAGGATTTGGAAATTGATTAGTACCTCT AATATTAGGTGCCCCTGATATAGCTTTCCCTCGAATAAATAA TATAAGTTTTTGACTCCTTCCCCCT TCTTTAACTCTTCTTATTTCGAGAAGAATTG TA??????????????????????????????????? ??????????????????????????????? ?????????????????????????????? ???????????????????????????????? ???????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ????????????????????????????????? ??????????????????????????????? ????????????????? Gindentata ??AACATTATATTTTATTTTTGGAATTTGAGCTGGTATAG TAGGAACCTCCTTAAGTTTATT AATTCGAGCTGAATTAGGT AATCCAGGATCTTTAATTGGAGATGATCAAATTTATAATAC TATTGTAACAGCACATGCTTTTATTATAATTTTTTTTAT AGTTATACCTATTATAATTGGAGGA TTTGGAAATTGATTAGTACCACTTATACTCGGAGCCCCTGATATAGCTTTCCCCC GAATAAATAACATAAGTTTCTGACTTCTTCCCCCATCTTT AACTCTTCTAATTTCAAGAAGTATTGTAGAAAGTGGAGCA

PAGE 199

GGAACAGGATGAACAGTTTAT CCCCCCCTTTCATCCAATATCGCTCATGGAG GTAGATCAGTTGATTTAGCTATTTTTTC ATTACACTTAGCTGGGATCTCATCAATTTTAGGAGCTATTAATTTCATTACAACAATTATTAA TATACGATTAAATAACT TATCATTTGATCAAATACCTTTATTTAT TTGAGCTGTAGGAATTACCGCATTCTTA CTACTTCTTTC TTTACCAGTTTTAG CAGGAGCTATTACTATACTTTTAAC TGATCGAAATTTAAATACCTCTTTTTTTG ATCCAGCAGGAGGAGGAGATCCTATT CTTTATCAACATTTATTT????? Gsinaldus ??AACATTATATTTTATTTTTGGAATTTGAGCTGGTATAGTA GGAACCTCTTTAAGTTTATT AATTCGAGCTGAATTAGGT AATCCTGGATCTTTAATTGGAGATGATCAAATTTATAATAC TATTGTAACAGCACATGCTTTTATTATAATTTTTTTCAT AGTTATACCTATTATAATTGGAGGATTT GGAAATTGATTAGTACCCCTTATATTAGGAGCACCTGATATAGCTTTCCCTC GAATAAACAATATAAGTTTTTGACTCCTTCCCCCTTCTTT AACTCTTTTAATTTCAAGAAGA ATTGTAGAAAGTGGAGCA GGAACAGGATGAACAGTTTACCCCCCA CTTTCATCTAATATTGCTCATGG AGGTAGTTCAGTTGACTTAGCTATTTTTTC CTTACATTTAGCTGGAATTTCATCAATT TTAGGAGCTATTAATTTCATTACAACAATTATTAATATACGATTAAATAATT TATCATTTGATCAAATACCTTTATTTATTTGAGCTGTAGGCATTACTGCATTCTTATTACTTCTCTCTTTACCGGTTTTAG CTGGAGCTATTACAATACTTT TAACTGACCGAAATTTAAA TACTTCTTTTTTTGACCCTG CAGGAGGAGGAGATCCTATT TTATATCAACACTTA TTC????? Gparens ?????????????ATTTTTGGTATTTGAGCTGGAAT AGTAGGAACATCTTTAAGATTATT AATTCGAGCTGAAT TAGGAAATCC TGGATCTTTAATTGGAGATGATCAAA TTTATAATACTATCGTAACAGCACAT GCTTTTATTATAATTTTTTTTATAGTAA TACCTATTATAATTGGAGGATTTGGAAATTGATTAGTACCT CTTATATTAGGAGCCCCTGA TATAGCTTTTCCTCGAATA AATAATATAAGTTTTTGACTCCTTCCCC CTTCTCTAACTCTTTTAATTTCAAG AAGAATTGTAGAA AGTGGAGCAGGAAC AGGATGAACAGTTTACCCCCCACTTTCA TCTAATATTGCTCATGGAGGAAGT TCAGTTGATTTAGCTATTTTTTCATTAC ATTTAGCAGGAATTTCATCAATTTTAGG AGCTATTAATTTTATTACAACAATTA TTAATATACGACTAAATAATTTATCA TTTGATCAAATACCTTTATTTATTTGAGCTGTAGGTATTACA GCATTTTTATTACTTCTTTCTTTACCAGTTTTAGCAGGA GCTATTACTATACTTTTAACTGATCGAAA TTTAAATACTTCATTTTTTGACCCAGCTGGAGGAGG??? ???????????????????? ???????????? 199 Gincurva ??AACATTATATTTTATTTTTGGTATT TGAGCTGGAATAGTAGGAACATCTTTAAGATTACTAATTCGAGCTGAATTAGG AAATCCTGGATCTTTAATTGGAGATGATCAAATTTATAATACT ATCGTAACAGCACATGCTTTTATTATAATTTTTTTTA TAGTAATACCTATTATAATTGGAGGATTC GGAAATTGATTAGTACCTCTTATA TTAGGAGCCCCCGATATAGCTTTTCCT CGAATAAATAATATAAGTTTCTGACTCCTTCCCCCTTCTC TAACTCTTTTAATTTCAAGAAG AATTGTAGAAAGCGGAGC AGGAACAGGATGAACAGTTTACCC CCCACTTTCATCTAATATTGCTCATGGAGG AAGTTCAGTTGATTTAGCTATTTTTT CACTTCATTTAGCAGGAATTTCATCAATTTTAGGAGCTATTAATTTTATTACA ACAATTATTAATATACGATTAAATAAT TTATCATTTGATCAAATA CCTTTATTTATTTGAGCTGTAGGTATTACAGCATTCTTATTA CTTCTTTCTTTACCAGTTTTA

PAGE 200

GCGGGAGCTATTACTATACTTTTAAC TGATCGAAATTTAAATACCTCATTTTTC GATCCAGCTGGAGGAGGTGATCCTAT TCTTTATCAACATTTATTT????? Gcorrecta ??AACATTATATTTTATTTTTGGTATT TGAGCTGGAATAGTAGGAACATCTTTAAG ATTATTAATTCGAGCTGAATTAGG AAATCCTGGATCTTTAATTGGAGATGATCAAATTTATAATACT ATCGTAACAGCACATGCTTTTATTATAATTTTTTTTA TAGTAATACCTATTATAATTGGAGGATTC GGAAATTGATTAGTACCTCTTATA TTAGGAGCCCCCGATATAGCTTTTCCT CGAATAAATAATATAAGTTTTTGACTCCTTCCCCCTTCTC TAACTCTTTTAATTTCAAGAAG AATTGTAGAAAGTGGAGC AGGAACAGGATGAACAGTTTACCC CCCACTTTCATCTAATATTGCTCATGGAGG AAGTTCAGTTGATTTAGCTATTTTTT CACTACATTTAGCAGGAATTTCATCAATTTTAGGAGCTATTAATTTTATTACAACAATTATCAATATACGATTAAATAAT TTATCATTTGATCAAATACCTTTATTTATTTGAGCTGTAGGTATTACAGCATTTTTATTACT TCTTTCTTTACCAGTTTTAG CAGGAGCTATTACTATACTTTTAACTG ATCGAAATTTAAATACTTCATTTTTTG ATCCAGCTGGAGG AGGTGACCCTATT CTTTATCAACATTTATTT????? Gsicheas ??AACATTATATTTTATTTTTGGAATTTGAGCTGGAATAGTAGGAACTTCTTTAAGTTTATT AATTCGAGCTGAATTAGGT AATCCTGGATCTTTAATTGGAGATGATCAAATTTATAATAC TATTGTTACAGCACATGCTTTTATTATAATTTTTTTTATA GTTATACCTATTATAATTGGAGGTTTTGGTAATTGATTAGTACCTCTTATACTCGGAGC TCCTGATATAGCTTTCCCCCG AATAAATAATATAAGTTTCTGACTTCT TCCCCCCTCTTTAACTCTTCTAATTTC AAGAAGAATTGTAGAAAGTGGAGCAG GAACAGGATGAACAGTTTACC CCCCACTTTCATCTAATATTG CTCATGGAGGAAGTTCAG TTGATTTAGCTATTTTCTCA TTACATTTAGCAGGAATTTCATCAATTTTAGGAGCTATTA ATTTTATCACCACAATTATTA ATATACGATTAAATAATTT ATCATTTGATCAAATACCTTTATTTGTTTGAGCTGTAGGTAT TACCGCATTTTTATTACTTCTTTCACTACCAGTTTTAGC AGGAGCTATTACTATACTTTTAACTGATCGAAATTTAAATAC TTCATTTTTTGACCCCGCAGGAGGAGGAGATCCTATTC TCTACCAACATTTATTT????? 200 Guxor ??AACATTATATTTTATTTTTGGTATT TGAGCTGGAATAGTAGGAACATCTTTAAGATTACTAATTCGAGCTGAATTAGG AAACCCTGGATCTTTAATTGGAGATGATCAAATTTATAATACT ATCGTAACAGCACATGCTTTTATTATAATTTTTTTTA TAGTAATACCTATTATAATTGGAGGATTC GGAAATTGATTAGTACCTCTTATA TTAGGAGCCCCCGATATAGCTTTTCCT CGAATAAATAATATAAGTTTTTGACTCCTTCCCCCTTCTC TAACTCTTTTAATTTCAAGAAG AATTGTAGAAAGTGGAGC AGGAACAGGATGAACAGTTTACCC CCCACTTTCATCTAATATTGCTCATGGAGG AAGTTCAGTTGATTTAGCTATTTTTT CACTACATTTAGCAGGAATTTCATCAATTTTAGGAGCTATTAATTTTATTACA ACAATTATCAACATACGATTAAATAAT TTATCATTTGATCAAATACCTTTATTTATTTGAGCTGTAGGTATTACAGCATTTTTATTACT TCTTTCTTTACCAGTTTTAG CGGGAGCTATTACTATACTTTTAACTG ATCGAAATTTAAATACTTCATTTTTCGA CCCAGCTGGAGGAGGTGATCCTATT CTTTATCAACATTTATTT?????

PAGE 201

Hsittaca ?????????????ATTTTTGGAATTTGAGCAGG AATAGTAGGAACTTCTTTAAGTTTATT AATTCGAGCTGAAT TAGGTAACCC CGGATCATTAATTGGAGATGATCAAATT TATAATACTATTGTTACAGCTCATGCTTTTATTATAATTT TTTTTATAGTTAT ACCTATTATAATTGGAGGATTTGGTAATTGATTAGTACCTTT AATATTAGGAGCTCCTGATATAGCTTTCCCTCGTATAA ATAATATAAGTTTTTGACTCTTACCCCCTTCTTTAACTCTTTTAATTTCT AGAAGAATTGTA????????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ?????????????????????????????????????????????????????????????????? ????????????????????? Oemarginata ?????????????ATTTTTGGTATTTGAGCTGGTATAGTAGGAACTTCTTTAAGATTATTAATTCGAGCTGAATTAGGAAATCC TGGTTCTTTAATTGGTGATGACCAAATTTATAACACTAT TGTAACAGCCCATGCTTTTATT ATAATTTTTTTTATAGTTAT ACCTATTATAATTGGAGGATTTGGAAACT GATTAGTTCCTCTAATATTAGGAGCAC CTGATATAGCTTTTCCTCGTATAA ATAATATAAGTTTTTGAC TTCTTCCACCTTCTTTAACCCTTTT AATTTCTAGAAGAATTGTA????? ???????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ???????????????????????????????????? ?????????????????????????????????????????????????????? ?????????????????????????????????????????????????????????????????? ????????????????????? Onobilis ??AACATTATATTTTATTTTTGGAATTTGAGCAGGTATAGTA GGAACATCTCTAAGATTATT AATTCGAGCTGAATTAGG TAATCCAGGATCTTTAATTGGAGATGATCAAATTTACAATACT ATTGTAACAGCTCATGCTTTTATTATAATTTTCTTTAT AGTTATACCTATTATAATTGGAGGTTTT GGAAATTGATTAGTACCTTTAATGTTAGGAGCACCTGATATAGCTTTCCCAC GAATAAATAATATAAGTTTCTGACTTCTTCCCCCTTCTTT AACTCTTCTAATTTCTAGAAGAATTGTAGAAAATGGAGCA GGAACTGGATGAACAGTTTACCCCCCACTTTCATCTAATATTGCCCATAGAGGT AGATCAGTGGATTTAGCTATTTTTTC ACTTCACTTAGCAGGAATCTCATCAATTTTAGGAGCAATTA ATTTTATTACTACAATTATTA ATATACGACTTAGAAATT TATCTTTTGATCAAATACCTTTATTTG TGTGAGCTGTAGGTATTACAGCCTTTTTA CTGCTATTATCTCTTCCTGTTTTAG CTGGAGCTATTACTATACTTTTAAC AGACCGAAATCTAAATACATCCTT TTTTGACCCAGCTGGAG GAGGAGATCCAAT CTTATATCAACATTTATTT????? 201 Orectristria ???????????TTATTTTTGGTATTTGAGCAGGtATAGTAGGAACCTCTTTA AGATTATTAATTCGAGC TGAATTAGGTAATCC AGGATCCTTAATTGGTGATGATCAAATCTATAATACTATTGTAACAGCTCATGCTTTTATTATAATTTTTTTCATAGTTAT ACCTATTATAATTGGAGGATTTGGAAATTGATTGGTTCCATTAATATTAGGAGCACCTGATATAGCTTTCCCACGTATAA ATAATATAAGTTTTTGACTTCTTCCTCC ATCATTAACTCTTTTAATTTCCAGAAG AATTGTAGAAAATGGAGCAGGAACT GGATGAACAGTCTATCCACCACTTTCATCAAATATTGCTCAC GGGGGAAGATCTGTTGATTTAGCCATTTTTTCTCTTCA TTTAGCTGGAATTTCATCAATTTTAGGAG CAATTAATTTTATTACAACAATTATT AATATACGACTAAATAATTTATCAT

PAGE 202

TTGATCAAATACCACTATTTGTATGAGCTGTTGGTATTACTGCTTTCTTACTTTTATT ATCTTTACCAGTTTTAGCAGGAG CTATTACAATATTATTAACTGATCGAAATTTAAATACATCATTTTTTGACCCTGC????????????? ????????????????????????? ?????? Oserpans ??AACATTATATTTTATTTTTGGTGTA TGAGCAGGTATAGTAG GAACATCTTTAAGATTATTAATTCGAGCTGAATTAGG TAATCCAGGATCTCTTATTGGAGATGATCAAATTTATAATACT ATTGTAACAGCACATGCTTTTATTATAATTTTTTTCAT AGTTATACCTATTATAATTGGAGGATTT GGAAATTGATTAGTTCCTTTAATATTAGGAGCCCCAGATATAGCTTTCCCTC GAATAAATAATATAAGTTTTTGACTTCTTCCCCCATCA TTAATTTTACTAATTTCAAGAAGAATTGTAGAAAATGGAGC AGGAACTGGATGAACAGTGTACCCCCCACTATCATCAAA TATTGCACACGGAGGAAGATCAGTAGATTTAGCTATTTTC TCCCTTCATTTAGCTGGAATTTCATC AATTTTAGGAGCTATTAATTTTATTACAACAATTATTAAT ATACGATTAAATAA TATATCATTTGATCAAATACCTTTATTTATTTGAGCTGTA GGAATTACAGCTTTCTTATTATTACTTTCTCTTCCTGTTTTA GCAGGAGCTATTACAATACTTTTAAC AGATCGAAATTTAAATACATCTTTTTTTGACCCAGCTGGTGGAGGAGATCCTA TTTTATATCAACATTTATTT????? Oexcitans ??AACATTATATTTTATCTTTGGTATTTGAGCAGGTATAG TAGGAACATCATTAAGATTATTAATTCGAGCTGAGTTAGG TAATCCTGGATCTCTTATTGGAGATGATCAAATTTATAATACCATTGTAACAGCTCATGCTTTTATTATAATTTTTTTCAT AGTTATACCTATTATAATTGGAGGA TTTGGAAATTGATTAGTTCCATTAATAT TAGGAGCCCCAGATATAGCTTTTCCTC GAATAAATAATATAAGTTTTTGACTTTTACCCCCCTCATT AACTCTTTTAATTTCAAGAA GAATTGTAGAAAACGGAGC AGGAACTGGATGAACAGTGTACCCCCCACTTTCATCTAATATTGCTCATGGA GGAAGATCTGTAGATTTAGCTATTTTTT CCCTTCATCTAGCTGGAATTTCTTCAATTTTAGGAGCTATTA ATTTTATTACAACAATTATC AATATACGATTAAATAAT TTATCATTTGACCAAATACCT TTATTTGTTTGAGCTGTTGGAA TTACAGCTTTCCTATTACTTC TATCCCTTCCTGTTTTA GCAGGAGCTATTACTATACTTTTA ACAGACCGAAATTTAAATACATCTT TTTTTGATCCAGCTGGT GGAGGAGACCCTA TTTTATATCAACATTTATTT????? 202 Pcoelonota ???????????????????????TCTGAGCAGGtAT AGTAGGAACTTCATTAAGATTACTAA TTCGAGCAGAATTAGGTAACCCTGG ATCTTTAATTGGAGATGATCAAATTTATAATACTATTGTCACAGCTCATGC TTTCATTATAATTTTTTTTATAGTTATACC TATTATAATTGGAGGATTTGGTAATTG ATTAGTTCCACTTATA TTAGGTGCACCTGATATAGCTTTCCCTCGTATAAATA ATATAAGTTTTTGACTCCTTCCCC CCTCTTTAACTCTTTTAATTTCCAGAAGAAT TGTAGAAAATGGAGCTGGAACAGGA TGAACAGTTTATCCCCCACTATCTTCTAATATTGCTCA CGGAGGTAGATCTGTAGATTTAGCTATTTTTTCATTACATTTA GCAGGAATTTCATCAATTT TAGGAGCTATTAATTTTATTA CAACAATTATTAATATACGATTAAATAATCTTTCATTTGA TATAATACCATTATTTGTATG AGCAGTAGGTATTACTGCATTCTTACTATTA TTATCACTCCCAGTCTTAGCTGGTGCCA TTACTATACTATTAACTGACCGAAATTTAAATACCTCTTT?????????? ??????????????????????????? ???????????????????

PAGE 203

Pcompressipalpus ??????????TTTATTTTTGGAATTTGAGCTGGTATAATTGGAACCTCATTAAGATTATTAATTCGAG CAGAATTAGGAAATC CTGGCTCTTTAATTGGAGATGA TCAAATTTATAATACTATTG TAACAGCCCATGCTTTCAT CATAATTTTTTTTATAGTTA TACCTATTATAATTGGAGGATTTGGAAATTGATTAGTACCTTTAATACTAGGAGCCCCTGATATAGCTTTCCCCCGAATA AACAACATAAGTTTCTGACTTC TTCCCCCTTCTTTAACTCTTTT AATTTCTAGAAGAATTGTA GAAAACGGAGCAGGAAC CGGCTGAACAGTTTACCCCCCTTTATCATCTAATATC GCACATAGTGGAAGATCTGTAGATTTAGCTATTTTTTCCCTAC ATTTAGCTGGAATCTCTTCCATTTTAGG AGCAATTAATTTTATTAC GACAATTATTAATATACGAT TAAATAATCTTTCA TTTGATATAATACCTTTATTTGTTTGAGCT GTAGGTATTACTGCATTTTTATTATT ACTATCTTTACCAGTATTAGCAGGA GCTATTACCATATTATTAACTGATCGTAATTTAAATACTTCTTTTTTCGACCCCG CTGGGGGAGGAG???????????????????? ????????????? Pdimorpha ?????????????ATTTTTGGAATTTGAGCCGGT AtAGTAGGAACTTCA TTAAGATTACTAATTCGAGCAGAATTAGGTAACCC CGGATCTTTAATTGGAGATGATCAAATT TATAATACTATTGTTACAGCTCATGCTTTTATTATAATTT TTTTTATAGTTAT ACCTATTATAATTGGAGGATTTGGAAATTGATTAGTTCCA TTAATATTAGGAGCCCCTGATATAGCTTTCCCTCGAATAA ATAATATAAGTTTTTGACTTTTACCCCCCTCTTTAACTC TTTTAATCTCCAGAAGAATCGTAGAAAATGGAGCTGGAACA GGATGAACAGTCTACCCCCCACTATCATCTAATATTGC CCATGGAGGTAGCTCTGTAGATTTAGCTATTTTTTCTTTACA TTTAGCTGGAATTTCTTCAATTTTAGGAG CAATTAATTTTATTACAACAATTATTAATATACGACTTAATAATCTTTCATT TGATATAATACCATTATTTGTATGAGCTGTAGGAATTACTGCATTTTTATTATTACTATCTCTTCCAG TTTTAGCCGGAGC TATTACTATATTATTAACTGATCGAAATTTA AATAC??????????????????? ??????????????????????????????????????????? 203 Slibatrix AACTTTATACTTTATTTTTGGTATTTG AGCTGGAATAGTAGGAACTTCTTTAAGT ATATTAATTCGAGCTGAATTAGGAA ATCCAGGATCATTAATTGGAAATGATCAAATTTATAATACTATTGTAACAGCGCACGCTTTT ATTATAATTTTTTtTATAG TTATACCTATTATAATTGGAG GATTTGGTAATTGATTAGT TCCTTTAATATTAGGAGCTCCTGATATAGCTTTCCCTCGA ATAAATAATATAAGTTTCTGACTTCTACCCC CTTCTTTAATTTTATTAACCTCAAG????????????????????????????????????? ???????????????????????????GCCCATA GAGGAAGATCAGTAGATCTTGCTATTTTTTCTCTTCATTTAGCAGGAATTTCTTC AATTTTAGGTGCTATTAATTTCATTACAACAATTATTAATATACGATTAAATAA TTTATCATTTGATCAAATACCTTTATT TGTTTGAGCTGTAGGGATCACAGCATTTTTATTATTATTATC TCTTCCAGTATTAGCAGGAGCTATTACAATACTTTTAA CTGATCGAAATTTAAATACTTCATTTTTTGACCCTGCAGGA GGAGGTGATCCAATTCTTTATCAACATTTATTT??????? Ccanadensis ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ?????????????????????????

PAGE 204

??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ???????????????????????????? ???????????????????????????????? Etyrannus ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????????????????????????????????????? ???????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ?????????????????????????????????????????????????????????????????? ?????????????????????????????????? ????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ???????????????????????????? ???????????????????????????????? Gnutrix ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ???????????????????????? ?????????????????????????????? ???????????????????????????????????? ???????????????????????????? ???????????????????????????????? Hhormos ??????????????????????????????? ?????????????????????????????? ???????????????????????????????? ???????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ????????????????????????????????????? ??????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ???????????????????????????????????????????????????????????? ???????????????????????????? ???????????????????????????????? 204 Hmonilis ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ???????????????????????? ?????????????????????????????? ???????????????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ???????????????????????????? ???????????????????????????????? Phycallitrichoides ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ????????????????????????????????????? ???????????????????????

PAGE 205

??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ?????????????????????????????????????????????????????????????????? ??????????????????????????????????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ???????????????????????????? ???????????????????????????????? Pcasta ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ???????????????????????????????????????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ???????????????????????????? ???????????????????????????????? [28S] Slibatrix ???TCGAATGAACGA??ACGGAGAGATTC ATCGTCATTCCGCGGCGT ACGCGATGCGCGACTCG ATGTCGT?CGGCCGAG GCC?GGCGTGCACGACGTGCGTCCGTC?ACGTCCGTGGACGGCGTGCACTTCT CTCTTAGTA?AATACATCGCGACCCGT TCGATGTCGGTCTAAGCGCCGTTCGGGAGCCCCAC?GGTGT ?CC???????GGCAACGT?ACA CCGCGGGACCGCGACGGTG GCCGACCGGCCGTCGGACGGTAGTTC TGATGAAACGCGCACGCGTTTTTAACGCGTCCGGCCCGACGCAAGTCAACGT CGTA??TCC?AACGT????????CTCGCCCA AGTGCGAACGTAGGTGCGGCGCGCCTGC ???TGTTGCCGCCGTGCAGTCTCGG ACTGTGCGCGTCTCC?GTCTGCGATGA TTCA?GTTTCGGGCACTCG???????????????????? ???????????????????????????????? ??????????????????????????????? ?????????????????????????????????????????????????????????????? ???????????????????????????? ??????????????????????????????? ?????????????????????????????? ???????????? 205 Pcallitrichoides AACTCGAATGAACGA??ACGGAGAGATTCATCGTCACTCCGCGGCGTACG?GACGCGCGTTTCGATGTCGC?CGGCCACG GTC?GGCAGGCACTGCGCGCGTACGTCGACGTCCGCGGACGGCGTGCACTTCTCTCTCAGTATCACAACATCGCGACCC GTTCGATGTCGGTCTAAGCGCCGTCC GGGAGCCCAGA?GGTGA?C G???????GGCGACCGCGCA CCTCTGGACCGAGACG GTGGCCGACCGGCCGTCGGACGGTAGTTCTGATGAAACGCGCACGCGTTTACAACGCGTCCGGCCCGACGCAAGTCAA CGTCGTA??TCC?CACGTAATTATATACCGCCTATGCGCGGA CGCGGGTGCGGCGCGTCTGC???CGTTGCAGCCGTGCAG TCTCGGACTGTGCGCGTCTCTCGTCTGCGATGATTCA?GTTTCGGGCA CTCGCAGGACCCGTCT TGAAACACGGACCAA GGAGTCTAGCATGTATGCGAGTCATTGAGATTTGATCAA AACTGAAAGGCGCAACGAAA GTGAAGGCGCGCGCTCGTC GCGCGCTCAGGGAGGATGGAGCCACGATCTAGGTCGTACT CTCGCACTCCCGAGGCGTCTCGTTTCCAATCCGTGAATG TAGGCGCGCTCTGAGCATA AATGCTGGGACCCGAAAGATGGT GAACTATG???????????

PAGE 206

Etyrannus ??????AATGAACGA??ACGGAGAGATTCATCGTCACTCCTCGGCGTACG?GGCGT GCGACTCGATGTCGTCCGGCTTCGGC CGGGCGCGCACGACGCGCGCTCGTCGACGCCCGGGGACGGCGTGCACTTCTCTCTTAGTA??AATGCATCGCGACCCGT TCGATGTCGGTCTAAGCGCCGTTCGGGAGCCCCGTGCGTGCGCTCTAATAAAGTCGCGC??????GCGGGACCGCGACGGT GGCCGACCGGCCGTCGGACGGTATCACTGACGAAGCGCGCA CGCGTTTACAACGCGTCCG GCCCGACGCAAGCCAACG TCGTATTTCC?GACGT????????ACCGCCAAAGT GCGGACGCCGGTGCGGCGTAGCTG T???CGTTGCTGCCGTGCAGTCTC GGACTGTGCGCGTCTCT?GTCTGCGATGATTCA?GTTTCG GGCACTCGCAGGACCCGTC TTGAAACACGGACCAAGGAG TCTAGCATGTATGCGAGTCATTGAGATT?CATTTAAACTGA AAGGCGCAACGAAAGTGAAGG CGCGCGCTTGCCGCGTG CTCAGGGAGGATGGAGCGTCG ATCTCGGTCGATCTCTCGCACTCCCGAGG CGTCTCGTTTCCAATCTGTGAATGCAGGC GCGCTCTGAGCATAAATGCTGGGACCCGAAAGATGGTGAACTATGCAT?GGTCAGA Guxor ??????AATGAACGA??ACGGAGAGATTCATCGTCATTCCGCGGCGTACG?CGCGCG CGCCTCGATGTCGT?CGGCCTCGGT C?GGCGTGCACGACGCGCGCGCGTCGACGTCCGCGGACGGCGTGCACTTCTCTCTTAGTA??TAAACATCGCGACCCGTT CGATGTCGGTCTAAGCGCCG TCCGGGAGCCCCGT? TGCGC?CT?????????TCGCGG?GCGC TTCGGGACCGCGACGGTGGC CGACCGGCCGTCGGACGGTAGTTTCGAA GAAACGCGCACGCGTTCACAACGCG TCCGGCCCGACGCAAGTCAACGTCG TA??TCC?AACGT???????CACCGCCTAAGCGC GGACGTAGGTGCGGCGCGTCTGT?? ?TGTTGCAGCCGTGCAGTCTCGGA CTGTGCGCGTCTCT?GTCTGCGATGATTCA?GTTTCGGGC ACTCGCAGGACCCGTCTTGAA ACACGGACCAAGGAGTCTA GCATGTATGCGAGTCATTGAGATAATA ???AAACTGAAAGGCGCAACGAAAGTGA AGGCGCGCGCTCGCCGCGCGCTCA GGGAGGATGGCGCTGCGATCTCGGTCG CACGCTCGCACTCCCGAGGCGTCTCG TTTCCAATCTGTGAATGCAGGCGCGC TCTGAGCATAAATGCTGGGACCCGAAAGATGGTGAACTATGCCT?GGTCAGA 206 Gnutrix ????????????CGA??ACGGAGAGATTCATCGTCATTCCGCGGCGT ACG?CGCGCGCGCCTCGATGT CGT?CGGCCT CGGTC?G GCGTGCACGACGCGCGCGCGTCGACGTCCGCGGACGGCGT GCACTTCTCTCTTAGTA??TAAACATCGCGACCCGTTCG ATGTCGGTCTAAGCGCCGTCCGGG AGCCCCGT?TGCGC?CT?????????TCGC GG?GCGCTTCGGGA CCGCGACGGTGGCCG ACCGGCCGTCGGACGGTAGTTTCGAAGAAACGCGCACGC GTTTACAACGCGTCCGGCCCGACGCAAGTCAACGTCGTA ??TCC?AACGT???????CACCG CCTAAGCGCGGACGTAGGTGC GGCGCGTCTGT???TGTTGCA GCCGTGCAGTCTCGGACT GTGCGCGTCTCT?GTCTGCGATGATTCA?GTTTCGGGCAC TCGCAGGACCCGTCTTGAAA CACGGACCAAGGAGTCTAGC ATGTATGCGAGTCATTGAGATAATA???AAACTGAAAGGCG CAACGAAAGTGAAGGCGCGCGCTCGCCGCGCGCTCAGG GAGGATGGCGCTGCGATCTCGGTCGCACGCTCGCACTCC CGAGGCGTCTCGTTTCCAATCTGTGAATGCAGGCGCGCTC TGAGCATAAATGCTGGG ACCCGAAAGATGGTGAAT ??????????????? Ccanadensis ???TCGAATGAACGA??ACGGAGAGATTC ATCGTCATTCCGCGGCGT ACGTAGCGCGCGACTCGAT GTCGT?CGGCCTCGG TC?GGCGCGCACGACGCGCGCTCGTCTACGTCCGCGGACGGCGTGCACTTCTCTCTTAGTA??AATACATCGCGACCCGT

PAGE 207

TCGATGTCGGTCTAAGCGCCGTTCGGGAGCCCCAC?GGTGC GCC?????????TCACGGCACAC CGTGGGACCGCGACGGTG GCCGACCGGCCGTCGGACGGTAGTTTTGACGAATCGCG CACGCGTTTACAACGCGTCCGGCCCGACGCAAGTCAACGC CGTA??TCCTTGCGT???????CATCGCCTCAG CGCGAACGTAGGTGCGGCGCGTCTGC???TGTTGCCGCCGTGCAGTCTCG GACTGTGCGCGTCTCT?GTCTGCGATGATTCA?GTTTCGGG CACTCGCAGGACCCGTCTTGAAACACGGACCAAGGAGTC TAGCATGTATGCGAGTCATTGAGATTTTATATAAACTGAA AGGCGCAACGAAAGTGAAGGCGCGCGCTCGCCGCGCGC TCAGGGAGGATGGAGCGTCGGTCTAGGTCGATCTCTCGCA CTCCCGAGGCGTCTCGTTTCCAATCTGTGAATGCAGGCG CGCTCTGAGCATAAATGCTGGGACCC GAAAGATGGTGAA???????????????? Oexcitans ???TCGAATGAACGA??ACGGAGAGATTC ATCGTCATTCCTCGGCGT ACG?GACGCGCGGTTAGATGTCGT?CGGCCTCGG TC?GGCTGGCACGACGCGCGCACGTCGACGTCCGGGGACGGCGTGCACTTCTCTCTTAGTA??AATACATCGCGACCCGT TCGATGTCGGTCTAAGCGCCGTTCGGGAGCCCCAT?CGTTC?CT?????????TC ACGG?GTTCGGT GGGACCGCGACGGTGG CCGACCGGCCGTCTGACGGTAGTTCTT AAGAAGCGCGCACGCGTTTACAACGCGTCCGGCCCGACGCAAGTCAACGTC GTA??TCC?TACGT????????ACCGCCTAAGCGC GGACGTGGGTGCGGCGC GTCTGCCGTTGTTGCT GCCGTGCAGTCTCGG ACTGTGCGCGTCTCT?GTCTGCGATGA TTCA?GTTTCGGGCACTCGCAGGACCCGTCTTGAA ACACGGACCAAGGAGTCT AGCATGTATGCGAGTCATTGAGATTATA???AAACTGAAAGGCGCAACGAAAGTGAAGGCGCGCGCTCGCCGCGTGCTC AGGGAGGATGGAGCGTCGATCTAGGTCGATCTCTCGCAC TCCCGAGGCGTCTCGTTTCCAATCAGTGAATGCAGGCGCG CTCTGAGCATAAATGCTGGGACCCGAAAGAT GGTGAACTATGCCTGGACAG?? 207 Orectristria AACTCGAATGAACGA??ACGGAGAGATTCATCGTCATTCCTCGGCGTACG?GACGCGCGGTTAGATGTCGT?CGGCCTCG GTC?GGCTGGCACGACGCGCGCACGTCGACGTCCGGGGAC GGCGTGCACTTCTCTCTTAGTA??AATACATCGCGACCCG TTCGATGTCGGTCTAAGCGCCGTTCGGG AGCCCCAT?TGTAC?CT?????????TTAC GG?GTTCGGTGGGACCGCGACGGTG GCCGACCGGCCGTCTGACGGTAGTTTTTAAGAAGCGCGCACGCGTTTACAACGCGTCCGGCCCGACGCAAGTCAACGT CGTA??TCC?TACGT????????ACC GCCTAAGCGCGGACGTGGGTGCGGCGCGTC TGTCGTTGTTGCTG CCGTGCAGTCTCG GACTGTGCGCGTCTCT?GTCTGCGATGATTCA?GTTTCGGG CACTCGCAGGACCCGTCTTGAAACACGGACCAAGGAGTC TAGCATGTATGCGAGTCATTGAGATAATA???AAACTGAAA GGCGCAACGAAAGTGAAGGCGCGCGCTTGCCGCGTGCT CAGGGAGGATGGAGCGTCGATCTAGGTCGATCTCTCGCA CTCCCGAGGCGTCTCGTTTCCAATCAGTGAATGTAGGCGC GCTCTGAGCATAAATGCTGGGACCCGAAAGATGGTGAACTATGCCCTGGTCAGA Clata AACTCGAATGAACGA??ACGGAGAGATTCATCGTCATTCCGCGGCGTACG?GACGCGCGCTTCGATGTCGT?CGGCCTCG GTC?GGCCGGCACGACGCGCGTACGTCGACGTCCGCGGACGGCGTGCACTTCTCTCTTAGTA??AATACATCGCGACCCG TTCGATGTCGGTCTAAGCGCCGTCCG GGAGCCCCAT?TGTGC?CT?????????TAAC GG?GTATAGTGGGAC CGCGACGGTG GCCGACCGGCCGTCGGACGGTAGTTCTGACGAAACGCG CACGCGTTTAAAACGCGTCCGGCCCGACGCAAGTCAACGT CGTA??TCC?CACGT????????ACC GCCTCAGCGCGGACGCGGGTGCGGCGCGTCT GC???TGTTGCCGCCGTGCAGTCTCGG

PAGE 208

ACTGTGCGCGTCTCT?GTCTGCGATGA TTCA?GTTTCGGGCACTCGCAGGACCCGTCTTGAA ACACGGACCAAGGAGTCT AGCATGTATGCGAGTCATTGAGACAATA???AAACTGAAAGGC GCAACGAAAGTGAAGGCGCGCGCTTGCCGCGCGCTC AGGGAGGATGGAGCGTCGATCTCGGTC GACCTCTCGCACTCCCGAGGCGTCTCGTTTCCAATCTGTGAATGCAGGCGCG CTCTGAGCATGAATGCTGG GACCCGAAAGATGGTGAACTATGCCC???????? Pcompressipalpus ?????GAATGAACGA??ACGGAGAGATTCATCG TCATTCCGCGGCGTACG?GTCGCG CGTTTCGATGTCGT?CGGCCTCGGT C?GGCTGGCACGACGCGCGTCCGTCGACGTCCGCGGACGGCGTGCACTTCTCTCTTAGTA??AATACATCGCGACCCGTT CGATGTCGGTCTAAGCGCCGTTCGGG AGCCCCAT?TGTAC?CT?????????TAACGG?GTATAGT GGGACCGCGACGGTGGC CGACCGGCCGTCGGACGGTAGTTCTGAA GAAACGCGCACGCGTTCTAAACGCG TCCGGCCCGACGCAAGTCAACGTCG TA??TCC?TACGT????????ACCGCCTAAGCGCGGACGTGGGTGCGGCGCGTCTGC???TGTTGCCGCCGTGCAGTCTCGGAC TGTGCGCGTCTCT?GTCTGCGATGATTCAGGTTTCGGGC ACTCGCAGGACCCGTCTTGAAACACGGACCAAGGAGTCTA GCATGTATGCGAGTCATTGAGATAATA???AAACTGAAAGGCGCAACGAAAGTGA AGGCGCGCGCTAGCCGCGCGCTCA GGGAGGATGGAGCGTCGGTCTAGGTCGATCTCTCGCACTC CCGAGGCGTCTCGTTTCCAAT CTGTGAATGTAGGCGCGC TCTGAGCATAAATGCTGGGACCCGAA AGATGGTGAACTATGC?????????? Pcasta ??????AATGAACGA??ACGGAGAGATTCATCGTCATTCCGCGGCGTACG?GTCGCGCGCTTCGATGTCGT?CGGCCTCGGT C?GGCTGGCACGACGCGCGTCCGTCGACGTCCGCGGACGGCGTGCACTTCTCTCTTAGTA??AATACATCGCGACCCGTT CGATGTCGGTCTAAGCGCCGTTCGGG AGCCCCAT?TGTAC?CT?????????TAACGG?GTATAGT GGGACCGCGACGGTGGC CGACCGGCCGTCGGACGGTAGTTCTG ACGAAACGCGCACGCGTTTATAACGC GTCCGGCCCGACGCAAGTCAACGTCG TA??TCC?TACGT????????ACCGCCTAAGCGCGGACGTGGGTGCGGCGCGTCTGC???T GTTGCTGCCGTGCAGTCTCGGAC TGTGCGCGTCTCT?GTCTGCGATGATTCAGGTTTCGGGC ACTCGCAGGACCCGTCTTGAAACACGGACCAAGGAGTCTA GCATGTATGCGAGTCATTGAGATAATA???AAACTGAAAGGCGCAACGAAAGT GAAGGCGCGCGCTTGCCGCGCGCTCA GGGAGGATGGAGCGTCGATCTAGGTCGATCTCTCGCACTC CCGAGGCGTCTCGTTTCCAAT CTGTGAATGTAGGCGCGC TCTGAGCATAAATGCTGGGACCCGAA AGATGGTGAA???????????????? 208 Hmonilis ???GGAAATCAGTGACGAAGGTGGGATC??TCGTC?TTCCTCGGCGTACG?GGCGCGCNCCTCGATGTCGT?CGGCCTCGG TC?GGCTGGCACGACGCGCGTTCGTCGACGTCCGCGGACGGCGTGCACTTCTCTCTTAGTA??AATACATCGCGACCCGT TCGATGTCGGTCTAAGCGCCGTTCGGGAGCCCCAT?TGTA C?CT?????????TCGCGG?GTAT AGTGGGACCGCGACGGTGG CCGACCGGCCGTCGGACGGTAGTTCTGACGAAACGCGCACGCGTTTATAACGC GTCCGGCCCGACGCATGTCAACGTC GTA??TCC?TGCGT????????ACCGCCTAAGCGC GGACGTAGGTGCGGCGCGTCTGT?? ?TGTTGCAGCCGTGCAGTCTCGGA CTGTGCGCGTCTCT?GTCTGCGATGATTCA?GTTTCGGGC ACTCGCAGGACCCGTCTTGAA ACACGGACCAAGGAGTCTA GCATGTATGCGAGTCATTGAGATAATA ???AAACTGAAAGGCGCAACGAAAGTGAAGGCGCGCCCTTGTCGCGCGCTCA

PAGE 209

GGGAGGATGGAGCGTCGATCTAGGTCGATCTCTCGCACTC CCGAGGCGTCTCGTTTCCAAT CCGTGAATGTAGGCGCGC TCTGAGCATAAATGCTGGGACCCGAAAGATGGTGAACTATGCCTGGGTCAGA Hhormos ??????AATGAACGA??ACGGAGAGATTCATCGTCATTCCTCGGCGTACG?GGCGCGCGCCTCGATGTCGT?CGGCCTCGGT C?GGCTGGCACGACGCGCGTTCGTCGACGTCCGCGGACGGCGTGCACTTCTCTCTTAGTA??AATACATCGCGACCCGTT CGATGTCGGTCTAAGCGCCGTTCGGG AGCCCCAT?TGTAC?CT?????????TCGCGG?GTATAGTGGGACCGCGACGGTGGC CGACCGGCCGTCGGACGGTAGTTCTG ACGAAACGCGCACGCGTTTATAACGC GTCCGGCCCGACGCAAGTCAACGTCG TA??TCC?TGCGT????????ACCGCCTAAGCGCGGACGTAGGTGCGGCGCGTCTGT???T GTTGCAGCCGTGCAGTCTCGGAC TGTGCGCGTCTCT?GTCTGCGATGATTCA?GTTTCGGGCACTCGCAGGACCCGTCTTG AAACACGGACCAAGGAGTCTAG CATGTATGCGAGTCATTGAGATAATA ???AAACTGAAAGGCGCAACGAAAGTGAAGGCGCGCCCTTGTCGCGCGCTCAG GGAGGATGGAGCGTCGATCTAGGTCGATCTCTCGCACTCC CGAGGCGTCTCGTTTCCAATCCGTGAATGTAGGCGCGCT CTGAGCATAAATGCTGGGACCCGAAAGAT GGTGAACTATGCCT?GGTCAGA Ematerna ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ???????????????????????????????? ?????????????????????????????????? ??????????????????????????????????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????????????????????????????????????? ???????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ?????????????????????????????? 209 Aflava ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ????????????????????????????????? ??????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ????????????????????????????????????? ??????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ???????????????????????????? ????????????????????????? ???????????????????????? ?????????????? Amesogona ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ???????????????????????????? ????????????????????????? ???????????????????????? ??????????????

PAGE 210

Calbivirgata ??????????????????????????????? ?????????????????????????????????????????????????????????????? ???????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????????????????????????????????????? ???????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????????????????????????????????????? ???????????????????????????? ???????????????????????????? ????????????????????????? ???????????????????????? ?????????????? Cbicolor ??????????????????????????????? ?????????????????????????????? ????????????????????????????????????? ??????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ???????????????????????????????????????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ???????????????????????????? ????????????????????????? ???????????????????????? ?????????????? Cthalictri ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ?????????????????????????????? 210 Eaurantia ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????????????????????????????????????? ???????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ????????????????????????????????? ??????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ???????????????????????????? ????????????????????????? ???????????????????????? ?????????????? Ejordani ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ?????????????????????????

PAGE 211

??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ???????????????????????????? ????????????????????????? ???????????????????????? ?????????????? Esalaminia ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ???????????????????????? ?????????????????????????????? ???????????????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ???????????????????????????????? ???????????????????????????? ???????????????????????????? ????????????????????????? ???????????????????????? ?????????????? Gindentata ??????????????????????????????? ?????????????????????????????????????????????????????????????? ???????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ?????????????????????????????????????????????????????????????????? ?????????????????????????????????? ????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ???????????????????????????????????????????????????????????? ???????????????????????????? ????????????????????????? ???????????????????????? ?????????????? Gsinaldus ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ???????????????????????????? ????????????????????????? ???????????????????????? ?????????????? 211 Gparens ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????????????????????????????????????? ???????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????????????????????????????????????? ???????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ?????????????????????????????? Gincurva ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ?????????????????????????

PAGE 212

??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ?????????????????????????????????????????????????????????????????? ??????????????????????????????????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ???????????????????????????? ????????????????????????? ???????????????????????? ?????????????? Gcorrecta ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ???????????????????????????? ????????????????????????? ???????????????????????? ?????????????? Gsicheas ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????????????????????????????????????? ???????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????????????????????????????????????? ???????????????????????????? ???????????????????????????? ????????????????????????? ???????????????????????? ?????????????? 212 Hsittaca ??????????????????????????????? ?????????????????????????????? ???????????????????????????????? ???????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ????????????????????????????????????? ??????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ???????????????????????????????????????????????????????????? ???????????????????????????? ????????????????????????? ???????????????????????? ?????????????? Oemarginata ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ???????????????????????????? ????????????????????????? ???????????????????????? ??????????????

PAGE 213

Onobilis ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????????????????????????????????????? ???????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????????????????????????????????????? ???????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ?????????????????????????????? Oserpans ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ???????????????????????????? ????????????????????????? ???????????????????????? ?????????????? Pcoelonota ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ???????????????????????? ?????????????????????????????? ???????????????????????????????????? ???????????????????????????? ????????????????????????? ???????????????????????? ?????????????? Pdimorpha ??????????????????????????????? ?????????????????????????????????????????????????????????????????????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ??????????????????????????????? ?????????????????????????????????????????????????????????????? ???????????????????????????? ??????????????????????????????? ?????????????????????????????? ??????????????????????????????????? ????????????????????????? ?????????????????????????????????????????????????????????????????? ?????????????????????????????????? ????????????????????? ???????????????????????????? ????????????????????????? ???????????????????????? ?????????????? ;END; 213

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BIOGRAPHICAL SKETCH Jennifer Michelle Zaspel was born in Nort h Branch, Minnesota. Upon graduation from North Branch High School in 1997, she began unde rgraduate studies at the University of Minnesota (Duluth). After transf erring to the University of Minnesota (Twin Cities) in 2000, she received her Bachelor of Science degree in scie nce in agriculture (with emphasis in entomology) in August of 2001. She then obtained a Master of Science degree in entomology from the University of Minnesota in 2004 under the advise ment of Dr. Susan Welle r. In August of 2004 she enrolled in a Ph.D. program at the University of Florida under the advi sement of Dr. Marc A. Branham in the Department of Entomology a nd Nematology. Her advisory committee members were Drs. Marjorie Hoy, Jacqueli ne Miller, and David Reed. Dr. Hans Bnziger served as an unofficial, ad hoc external committee member. Jennifer is currently a member of Sigma Xi Scientific Research Society, the Entomological Society of America, the Florida Entomolgical Society, the Lepidopterists Society, the Willi He nnig Society, the Society of Systematic Biologists, the American Association for the Advancement of Science (AAAS), and is on the Board of Directors for the Cent er of Systematic Entomology. 239