Universidad Austral De Chile Facultad De Ciencias Agrarias Escuela De Graduados

UNIVERSIDAD AUSTRAL DE CHILE FACULTAD DE CIENCIAS AGRARIAS ESCUELA DE GRADUADOS

DESCRIPCIÓN DE LARVAS DEL ENSAMBLE DE ESCARABAEIDOS PRESENTES EN PRADERAS Y ROL DE FACTORES DEL SUELO Y PLANTAS EN EL COMPORTAMIENTO Y DESARROLLO DE DOS ESPECIES NATIVAS.

TESIS DOCTORAL

ERNESTO SEGUNDO CISTERNAS ARANCIBIA

VALDIVIA - CHILE

2011

DESCRIPCIÓN DE LARVAS DEL ENSAMBLE DE ESCARABAEIDOS PRESENTES EN PRADERAS Y ROL DE FACTORES DEL SUELO Y PLANTAS EN EL COMPORTAMIENTO Y DESARROLLO DE DOS ESPECIES NATIVAS.

Tesis presentada a la Facultad de Ciencias Agrarias de la Universidad Austral de Chile en cumplimiento parcial de los requisitos para optar al grado de Doctor en Ciencias Agrarias.

por

ERNESTO SEGUNDO CISTERNAS ARANCIBIA

Valdivia - Chile

2011

Un buen caminante no deja huellas. / Un buen orador no se equivoca ni ofende. / Un buen contable no necesita útiles de cálculo. / Un buen cerrajero no usa barrotes ni cerrojos, y nadie puede abrir lo que ha cerrado. / Quien ata bien no utiliza cuerdas ni nudos y nadie puede desatar lo que ha atado…

Tao-Te-King, LaoTse

ii INDICE DE CONTENIDOS

Cap. Pag. 1. INTRODUCCIÓN GENERAL 1

2. DESCRIPCIÓN TAXONÓMICA DEL ESTADO LARVAL DE 13 DOS ESPECIES DE SCARABAEIDAE: MELOLONTHINAE: TRIBU INSERTAE SEDIS.

DESCRIPTION OF THE LARVAE OF Phytholaema herrmanni 14 Germain AND Phytholaema dilutipes (Fairmaire and Germain) (COLEOPTERA: SCARABAEIDAE: MELOLONTHINAE

ABSTRACT 14 RESUMEN 15 INTRODUCTION 15 MATERIAL AND METHODS 16 RESULTS 17 DISCUSSION 21 ACKNOWLEDGEMENTS 21 REFERENCES CITED 21

3 DESCRIPCIÓN TAXONÓMICA DEL ESTADO LARVAL DE 27 DOS ESPECIES DE SCARABAEIDAE: MELOLONTHINAE: MACRODACTYLINI

DESCRIPTION OF THIRD INSTAR OF Pristerophora picipennis 28 (Solier) AND Schizochelus modestus Philippi FROM SOUTHERN CHILE (COLEOPTERA: SCARABAEIDAE: MELOLONTHINAE: MACRODACTYLINI).

ABSTRACT 28 RESUMEN 29 INTRODUCTION 29 MATERIAL AND METHODS 30 RESULTS 31 ACKNOWLEDGEMENTS 35 REFERENCES CITED 36

DESCRIPCIÓN TAXONÓMICA DEL ESTADO LARVAL DE 4. UNA ESPECIE DE SCARABAEIDAE: MELOLONTHINAE: 42 LICHNIINI .

iii DESCRIPTION OF THE THIRD INSTAR OF Arctodim mahdii 43 Hawkins (COLEOPTERA: SCARABAEIDAE: MELOLONTHINAE: LICHNIINI).

ABSTRACT 43 RESUMEN 43 INTRODUCTION 44 MATERIAL AND METHODS 45 RESULTS 46 ACKNOWLEDGEMENTS 48 REFERENCES CITED 48

5. . DESCRIPCIÓN TAXONÓMICA DEL ESTADO LARVAL DE 52 DOS ESPECIES DE SCARABAEIDAE: RUTELINAE: ANOPLOGNATHINI: BRACHYSTERNINA.

DESCRIPTION OF THE LARVAE OF Hylamorpha elegans 53 Burmeister, 1844 AND Aulacopalpus punctatus (Fairmaire and Germain, 1860) (COLEOPTERA: SCARABAEIDAE: RUTELINAE: ANOPLOGNATHINI.

ABSTRACT 53 RESUMEN 53 INTRODUCTION 54 MATERIAL AND METHODS 55 RESULTS 56 DISCUSSION 60 ACKNOWLEDGEMENTS 61 REFERENCES CITED 62

6. SELECCIÓN DEL SITIO DE OVIPOSICIÓN DE Hylamorpha 68 elegans (Burm.) (SCARABAEIDAE: RUTELINAE).

OVIPOSITION SITE SELECTION BY THE SCARABAEID 69 BEETLE Hylamorpha elegans (Burm.) (COLEOPTERA: SCARABAEIDAE: RUTELINAE), IN RESPONSE TO PLANTS , SOIL TYPE AND COVERS

ABSTRACT 69 INTRODUCTION 70 MATERIAL AND METHODS 71 RESULTS 75 DISCUSSION 76

iv ACKNOWLEDGEMENTS 79 REFERENCES 80

7. SELECCIÓN DEL SITIO DE OVIPOSICIÓN DE Phytholaema 92 herrmanni Germain (SCARABAEIDAE: MELOLONTHINAE).

ENVIRONMENTAL FACTORS THAT DETERMINE THE 93 SELECTION OF OVIPOSITION SITE OF THE POLYPHAGOUS SCARABAEID Phytholaema herrmanni Germain (COLEOPTERA: SCARABAEIDAE: MELOLONTHINAE)

ABSTRACT 93 INTRODUCTION 94 MATERIAL AND METHODS 96 RESULTS 99 DISCUSSION 101 ACKNOWLEDGEMENTS 105 REFERENCES 106

8. EFECTO DEL TIPO DE SUELO Y LAS RAICES SOBRE EL 118 CRECIMIENTO Y DESARROLLO DE LAS LARVAS. Hylamorpha elegans (Burm.) (COLEOPTERA: SCARABAEIDAE: RUTELINAE).

GROWTH AND DEVELOPEMENT OF Hylamorpha elegans 119 Burmeister RUTELINAE (COLEOPTERA: SCARABAEIDAE) IN DIFFERENT SOIL TYPES WITH AND WITHOUT ROOTS.

ABSTRACT 119 INTRODUCTION 119 MATERIAL AND METHODS 121 RESULTS 124 DISCUSSION 126 ACKNOWLEDGEMENTS 129 REFERENCES CITED 129

9. EFECTO DEL TIPO DE SUELO Y LAS RAICES SOBRE EL 149 CRECIMIENTO Y DESARROLLO DE LAS LARVAS. Phytolaema herrmanni Germain (COLEOPTERA: SCARABAEIDAE: MELOLONTHINAE).

FITNESS OF Phytholaema herrmanni Germain (COLEOPTERA: SCARABAEIDAE: MELOLONTHINAE) LARVAE, REARED IN

v DIFFERENT SOIL TYPES WITH AND WITHOUT ROOTS 150 ABSTRACT 150 INTRODUCTION 151 MATERIAL AND METHODS 152 RESULTS 155 DISCUSSION 158 RESUMEN 162 CONCLUSIONS 162 ACKNOWLEDGEMENTS 163 LITERATURE CITED 164

10. DISCUSIÓN GENERAL 184 BIBLIOGRAFIA 191

ANEXOS 201

vi INDICE DE CUADROS

Pag. CAPITULO 1

Cuadro 1. Posición sistemática de las especies de scarabaeidos que se describen 6 en esta tesis de acuerdo a Evans y Smith, 2009.

CAPITULO 6

Table 1. Soil texture of the tested soils 84

CAPITULO 7

Table 1. Soil texture of the tested soils 110

CAPITULO 8

Table 1. Chemical characteristics of the soils used. 134 Table 2. Particulate matter content of organic matter (POM) greater than 53 µm 135 in the studied soils. Table 3. Basal respiration of the soils (CO2 mg. h-1.kg dry soil-1). 136 Table 4. Factorial analysis of variance (ANOVA) with repeated measurements 137 for larval live weight per instar of H. elegans. Table 5. Average weight per larval stage of H. elegans in three soil types, with 138 and without roots.

CAPITULO 9

Table 1. Chemical characteristics of the soils used. 169 Table 2. Particulate matter content of organic matter (POM) greater than 53 µm 170 in the studied soils. Table 3. Basal respiration of the soils (CO2 mg. h-1.kg dry soil-1). 171 Table 4. Factorial analysis of variance (ANOVA) with repeated measurements 172

vii for larval live weight per instar of P. herrmanni. Table 5. Average weight per larval stage of P. herrmanni in three soil types, 173 with and without roots.

viii INDICE DE FIGURAS

Pag. CAPITULO 2

Figs. 1-8. Phytholaema herrmanni, third instar. 1) Head, frontal view, distal 23 antennomere dorsal view; 2) Distal antennomere lateral view; 3) Epipharynx; 4) Mandibles, ventral view a) left and b) right; 5) Mandibles, dorsal view a) right and b) left; 6) Maxilla, a) dorsal view and b) ventral view; 7) Apex of lacinia and galea showing unci; 8) Detail of maxillary stridulatory area. Bar = mm. Figs. 9-15. Phytholaema herrmanni, third instar. 9) Labium dorsal view; 10) 24 Hypopharynx; 11) Thoracic spiracle; 12) Respiratory plate; 13) Tarsungulus of a) prothoracic leg, b) mesothoracic leg, and c) metathoracic leg; 14) Sclerotized vestigial structure on A9; 15) Last abdominal segment, ventral view of raster. Bar = mm. Figs. 16-23. Phytholaema dilutipes, third instar. 16) Head, frontal view, distal 25 antennomere dorsal view; 17) Distal antennomere, a) ventral view, b) lateral view; 18) Epipharynx; 19) Mandibles, ventral view a) left and b) right; 20) Mandibles, dorsal view a) right and b) left; 21) Maxilla, a) dorsal and b) ventral view; 22) Apex of lacinia and galea showing unci; 23) Detail of maxillary stridulatory area. Bar = mm. Figs. 24-30. Phytholaema dilutipes, third instar. 24) Labium, dorsal view; 25) 26 Hypopharynx; 26) Thoracic spiracle; 27) Respiratory plate; 28) Tarsungulus of a) prothoracic leg, b) mesothoracic leg, and c) metathoracic leg; 29) Sclerotized vestigial structure on A9; 30) Last abdominal segment, ventral view of raster. Bar = mm.

CAPITULO 3

Figs. 1-8. Pristerophora picipennis, third instar. 1) Head, frontal view, distal 38 antennomere dorsal view; 2) Distal antennomere lateral view; 3) Epipharynx; 4)

ix Mandibles, ventral view a) left, b) right; 5) Mandibles, dorsal view a) right, b) left; 6) Maxilla a) dorsal view, b) ventral view; 7) Apex of lacinia and galea showing unci; 8) Detail of maxillary stridulatory area. Bar = mm. Figs. 9-14. Pristerophora picipennis, third instar. 9) Labium dorsal view; 10) 39 Hypopharynx; 11) Thoracic spiracle; 12) Respiratory plate; 13) Tarsungulus of a) prothoracic leg; b) mesothoracic leg; c) metathoracic leg 14) Last abdominal segment, ventral view with raster. Bar = mm. Figs. 15- 22. Schizochelus modestus, third instar. 15) Head, frontal view, 16) 40 Distal antennomere ,a) dorsal ; b) ventral ;and c) lateral view; 17) Epipharynx; 18) Mandibles, ventral view, a) left and b) right; 19) Mandibles, dorsal view, a) right and b) left; 20) Maxillae, a) dorsal view and b) ventral view; 21) Apex of lacinia and galea showing unci; 22) Maxillary stridulatory area. Bar = mm. Figs. 23-28. Schizochelus modestus, third instar. 23) Labium dorsal view; 24) 41 Hypopharynx; 25) Thoracic spiracle; 26) Respiratory plate; 27) Tarsungulus of a) prothoracic leg, b) mesothoracic leg, c) metathoracic leg; 28) Last abdominal segment, ventral view of raster. Bar = mm.

CAPITULO 4

Figs. 1-8. Arctodium mahdii, third instar. 1) Head, frontal view; 2) Distal 50 antennomere, a) lateral and b) ventral view; 3) Epipharynx; 4) Mandibles, ventral view a) left and b) right; 5) Mandibles, dorsal view a) right and b) left; 6) Maxillae, a) dorsal, b) ventral and c) lateral view; 7) Apex of lacinia and galea showing unci; 8) Maxillary stridulatory area. Bar = mm. Figs. 9-15. Arctodium mahdii, third instar. 9) Labium, dorsal view; 10)

Hypopharynx; 11) Thoracic spiracle; 12) Abdominal spiracle A1; 13)

Respiratory plate; 14) Tarsungulus of legs a) prothoracic leg, b) mesothoracic 51 leg, c) metathoracic leg, 15) Last abdominal segment, ventral view of raster. Bar = mm

x CAPITULO 5

Figs. 1-8. Hylamorpha elegans, third instar. 1) Head, frontal view, distal 64 antennomere dorsal view ; 2) Distal antennomere lateral view; 3) Epipharynx; 4) Mandibles, ventral view, a) left and b) right; 5) Mandibles, dorsal view, a) right and b) left); 6) Maxillae, a) dorsal view and b) ventral view; 7) Apex of mala showing unci; 8) Maxillary stridulatory area. Bar = mm. Figs. 9-15. Hylamorpha elegans, third instar. 9) Labium dorsal view; 10) 65 Hypopharynx; 11) Thoracic spiracle; 12) Respiratory plate; 13) Tarsungulus of a) prothoracic leg, b) mesothoracic leg and c) metathoracic leg 14) Last abdominal segment, ventral view of raster. Bar = mm. Figs.15- 22. Aulocopalpus punctatus, third instar. 15) Head, frontal view; 16) 66 Distal antennomere, a) ventral , b) dorsal and c) lateral view; 17) Epipharynx; 18) Mandibles, ventral view a) left t and b) righ; 19) Mandibles, dorsal view, a) right and b) left ;20) Maxillae, a) dorsal and b) ventral view; 21) Apex of mala showing unci; 22) Maxillary stridulatory area. Bar = mm. Figs.15- 22. Aulocopalpus punctatus, third instar. 15) Head, frontal view; 16) 67 Distal antennomere, a) ventral , b) dorsal and c) lateral view; 17) Epipharynx; 18) Mandibles, ventral view a) left t and b) righ; 19) Mandibles, dorsal view, a) right and b) left ;20) Maxillae, a) dorsal and b) ventral view; 21) Apex of mala showing unci; 22) Maxillary stridulatory area. Bar = mm.

CAPITULO 6

Figure 1. Roots location in the soil cover experiments. 85 Figure 2. Oviposition preference of H. elegans for different soil types, sand, 86 sawdust and plants average for two seasons. Figure 3. Effect of soil covers and roots on the oviposition site selection of H. 87 elegans. First season. Figure 4. Effect of soil covers and roots on the oviposition site selection of H. 88

xi elegans. Second season. Figure 5. Effect of different densities of dummy plants on H. elegans 89 oviposition site selection. Figure 6. Effect of biomass dosage on the ovipositon selection site behavior of 90 H. elegans. Figure 7. Effect of visual and chemical cues on the selection of oviposition sites 91 by female H. elegans. WR: Without roots; WF: Without foliage; R: Roots; F: Foliage

CAPITULO 7

Figure 1. Roots location in the soil cover experiments. 111 Figure 2. Oviposition preference of P. herrmanni for different soil types, sand, 112 sawdust and plants average for two seasons. Figure 3. Effect of soil covers and roots on the oviposition behavior of P. 113 herrmann: first season. Figure 4. Effect of soil covers and roots on the oviposition site selection of P. 114 herrmanni. Second season Figure 5. Effect of different densities of dummy plants on P. herrmanni 115 oviposition site selection. Figure 6. Effect of the quantity of roots on the oviposition site selection 116 behavior of P. herrmanni. Figure 7. Effect of visual and chemical cues on the selection of oviposition sites 117 by female P. herrmanni. WR: Without roots; WF: Without foliage; R: Roots; F: Foliage

CAPITULO 8

Figure 1. Cumulative percentage survival of H. elegans larvae bred in three 139 soils, with and without wheat roots. (I: Inceptisol; IR: Inceptisol roots; A:

xii Andisol; AR: Andisol roots; U: Ultisol; UR: Ultisol roots). Figure 2: Relative growth rates (RGR) of H. elegans larvae in an inceptisol type 140 soil, with and without wheat roots. Figure 3: Relative growth rates (RGR) of H. elegans larvae in an andisol type 141 soil, with and without wheat roots. Figure 4: Relative growth rates (RGR) of H. elegans larvae in an ultisol type 142 soil, with and without wheat roots. Figure 5: Dynamic of the growth of H. elegans per instar in an inceptisol soil, 143 with and without wheat roots. Figure 6: Dynamic of the growth of H. elegans per instar in an andisol soil, with 144 and without wheat roots. Figure 7: Dynamic of the growth of H. elegans per instar in an ultisol soil, with 145 and without wheat roots. Figure 8: Larval stage development of H. elegans in an inceptisol type soil, with 146 and without wheat roots. A: without roots; B: with roots. Figure 9: Larval stage development of H. elegans in an andisol type soil, with 147 and without wheat roots A: without roots; B: with roots. Figure 10. Larval stage development of H. elegans in an ultisol type soil, with 148 and without wheat roots. A: without roots; B: with roots.

CAPITULO 9

Figure 1. Survival of P. herrmanni larvae bred in three soils, with and without 174 wheat roots. (I: Inceptisol; IR: Inceptisol roots; A: Andisol; AR: Andisol roots; U:Unltisol; UR: Ultisol roots). Figure 2: Relative growth rates (RGR) of P. herrmanni larvae in an inceptisol 175 type soil, with and without wheat roots. Figure 3: Relative growth rates (RGR) of P. herrmanni larvae in an andisol type 176 soil, with and without wheat roots.

xiii Figure 4: Relative growth rates (RGR) of P. herrmanni larvae in an ultisol type 177 soil, with and without wheat roots. Figure 5: Dynamic of the growth of P. herrmanni per instar in an inceptisol soil, 178 with and without wheat roots. Figure 6: Dynamic of the growth of P. herrmanni per instar in an andisol soil, 179 with and without wheat roots. Figure 7: Dynamic of the growth of P. herrmanni per instar in an ultisol soil, 180 with and without wheat roots. Figure 8: Larval stage development of P. herrmanni in an inceptisol type soil, 181 with and without wheat roots A: without roots; B: with roots. Figure 9: Larval stage development of P. herrmanni in an andisol type soil, with 182 and without wheat root A: without roots; B: with roots. Figure 10. Larval stage development of P. herrmanni in an ultisol type soil, with 183 and without wheat roots A: without roots; B: with roots.

xiv ANEXOS

Pag. ANEXO 1. 202 ESTRUCTURAS ESQUEMÁTICAS DE IMPORTANCIA TAXONÓMICA PARA LA DESCRIPCIÓN DE LARVAS DE ESCARABAEIDOS. (A) FORMA LARVAL; (B) CABEZA; (C) CUARTO ANTENITO; (D) EPIFARINGE .

ANEXO 2. 203 ESTRUCTURAS ESQUEMÁTICAS DE IMPORTANCIA TAXONÓMICA PARA LA DESCRIPCIÓN DE LARVAS DE ESCARABAEIDOS. (A) MANDÍBULA VISTA DORSAL; (B) MANDÍBULA VISTA LATERAL Y (C) MANDÍBULA VISTA VENTRAL.

ANEXO 3. 204 ESTRUCTURAS ESQUEMÁTICAS DE IMPORTANCIA TAXONÓMICA PARA LA DESCRIPCIÓN DE LARVAS DE ESCARABAEIDOS. (A) MAXILA Y LABIO DORSAL; (B) MAXILA LABIO VENTRAL; (C) DIENTES MAXILARES ESTRIDULADORES Y (D) UNCUS DE LA MAXILA.

ANEXO 4. 205 ESTRUCTURAS ESQUEMÁTICAS DE IMPORTANCIA TAXONÓMICA PARA LA DESCRIPCIÓN DE LARVAS DE ESCARABAEIDOS. (A) TERGO ABDOMINAL; (B) ESPIRÁCULO TORÁXICO; (C) PATA Y UÑA Y (D) RÁSTER.

ANEXO 5. 206 LISTA DE ABREVIATURAS DE ESTRUCTURAS TAXONÓMICAS

ANEXO 6. 207 DESCRIPCIÓN DE LAS ESTRUCTURAS DE IMPORTANCIA TAXONÓMICA PARA LA DESCRIPCIÓN DE LARVAS DE ESCARABAEIDOS.

1. INTRODUCCIÓN GENERAL

En el sur de Chile, las larvas y los adultos de los escarabaeidos son importantes componentes de las comunidades de insectos en sistemas intervenidos y naturales, produciendo principalmente los primeros daños significativos en praderas y cultivos anuales y plantaciones de frutales menores y especies forestales nativas (Durán, 1952; Durán, 1954; Durán, 1976; Prado, 1991; Klein y Waterhouse, 2000). La familia Scarabaeidae presenta un gran número de especies en todo el mundo, muchas de las cuales alcanzan altas densidades poblacionales y las transforman en importantes plagas, (Mannion et al., 2001). Entre las especies más importantes para Chile están: Hylamorpha elegans (Burm.), Phytholaema herrmanni Germain, Sericoides germaini Dalla Torre, Tamarus villosus Burm., Athlia plebeja Burm., Athlia rustica Erichson, Brachysternus prasinus Guérin-Méneville, Schizochelus serratus Philippi, Schizochelus breviventris Philippi, Sericoides viridis Solier , Sericoides obesa Germain y Aulacopalpus punctatus (Fairmaire and Germain) (Duran, 1963; Duran, 1964; Carrillo, 1986; Norambuena y Aguilera, 1988; Cisternas y Carrillo, 1989; Aguilera et al.,1996; Cisternas et al., 2000; Cisternas y Carrillo, 2001). Su biología, características taxonómicas y duración de sus ciclos de vida estacional y ontogenia; la selección del sitio de ovipostura, el crecimiento y desarrollo larval y pupación, la alimentación larval, herbivoría de raíces y uso de la materia orgánica del suelo como fuente de nutrientes, el apareamiento y alimentación de los adultos sobre el suelo corresponden a características singulares de muchos escarabaeidos (Duran, 1952; Durán, 1954; Cisternas y Carrillo, 1989; Brown y Gange, 1990; Aguilera et al., 1996; Potter y Held, 2002; Ward y Rogers, 2007; Harano et al.,2010 ).

Actualmente existe desconocimiento de las características morfológicas del estado larval de casi la totalidad de las especies de escarabaeidos en Chile, por lo que se hace necesario realizar estudios para la descripción de ellas y avanzar en la investigación sobre la biología y ecología de los mismos. En el último tiempo la taxonomía y sistemática de los imagos de

especies chilenas, ha sido revisada por Jameson y Smith, (2002); Smith, (2002); Ratcliffe y Ocampo, (2002), Hawkins, (2006), Smith, (2008), Mondaca, (2008), Evans y Smith, (2009), sin embargo existe aún un gran vacío en la descripción de los estados larvales de muchas de las especies que conforman los ensambles de escarabaeidos en el suelo.

Estructuras de importancia taxonómica en larvas de escarabaeidos

Las estructuras utilizadas para la descripción morfológica de las larvas de escarabaeidos son: cabeza, antena (dorsal, ventral y lateral), epifaringe, mandíbulas (dorsal, ventral y área estriduladora), maxila (dorsal, ventral), labio (dorsal, ventral), espiráculo toráxico, tergo abdominal, espiráculos abdominales y ráster (ventral, dorsal y lateral). Anexos 1 a 6.

Las larvas del tercer estadio son las utilizadas para realizar las descripciones de las especies y los términos taxonómicos usados para esta finalidad han sido descritos y empleados por Hayes (1929); Peterson (1960); Ritcher (1966); Edmonds y Halffter (1978); Vanin y Costa (1980); McQuillan (1985); Paucar-Cabrera y Smith (2002); Mico et al. (2003); Mico y Galante (2003); Grebennikov y Scholtz (2004) y Vallejo y Moron (2008).

Sistemática de las especies estudiadas

La familia Scarabaeidae agrupa al mayor número y diversidad de especies (27000) de la superfamilia Scarabaeoidea la que se estima posee unas 35000 especies alrededor del mundo agrupadas en catorce familias: Lucanidae, Passalidae, Trogidae, Glaresidae, Pleocomidae, Bolboceratidae, Diphyllostomatidae, Geotrupidae; Belohinidae, Ochodaeidae, Ceratocanthidae, Hybosoridae, Glaphyridae y Scarabaeidae. Al Neotrópico pertenecerian unas 6000 especies, Grebennnikov y Scholtz, (2004).

En la familia Scarabaeidae se reconocen trece subfamilias: Aphodiinae, Scarabaeinae, Pachypodinae, Orphninae, Allidiostomatinae, Dynamopodinae, Acoplinae, Euchirinae,

Phaenomeridinae, Melolonthinae, Rutelinae, Dynastinae y Cetoniinae, (Grebennnikov y Scholtz, 2004).

Subfamilia Melolonthinae Leach, 1819. Los Melolonthinae han sido agrupados en las siguientes tribus Pachypodini, Lichniini, Euchirini, Systellopini, Chasmatopterini, Oncerini, Podolasiini, Ablaberini, Sericini, Phyllotocidiini, Diphucephalini, Comophorinini, Stethaspini, Automoliini, Maechdiini, Liparetrini, Scitalini, Pachytrichini, Sericoidini, Heteronychini, Diplotaxini, Melolonthini, Pachydemini, Macrodactylini, Diphycerini, Hopliini, Colymbomorphini, Tanyproctini. (Smith, 2006; Evans y Smith 2009).

Tribu Incertae sedis. La posición tribal de Phytholaema Blanchard necesita más investigación para clasificar este género (Evans y Smith 2009). Se encuentra en la región Netropical y incluye a tres especies Phytholaema dilutipes (Fairmaire y Germain), Phytholaema herrmanni Germain, con dos subespecies P. herrmanni herrmanni Germain y P. herrmanni pallida Saylor y Phytholaema mutabilis (Solier) (Evans y Smith 2009). Todas estas especies son encontradas en Chile, solo P. mutabilis es también encontrada en Argentina, de acuerdo a Evans y Smith (2009). Según Smith (2006) la tribu válida a la cual pertenecería este género sería Stethaspini, sinonimia de Xylonichini en la que Britton (1957) y Mondaca (2008) adscriben al género Phytholaema. Esta tribu estaría sólo representada en Australia, Nueva Zelanda y Chile y correspondería a una tribu de origen Antártico según Britton (1957). Los adultos son conocidos como “San Juanes”, “Pololos” o “Pololo café chico” y las larvas en todos sus estadios como “gusanos blancos”. La morfología de las larvas de estas especies es desconocida. A pesar de la carencia de esta información, las larvas de estos melolontinos son considerados una parte importante de los ensambles de escarabaeidos en las praderas de la zona central y sur de Chile. El género Phytholaema es importante en los ecosistemas agrícolas, alcanzando sus larvas altas densidades, las que se alimentan de las raíces de un amplio rango de plantas, causando considerables daños económicos en plantas pratenses y cultivadas (Durán, 1954).

Tribu Macrodactylini Kirby, 1837. A esta tribu pertenecen las especies P. picipennnis (Solier) y Sch. modestus (Philippi). El género Schizochelus Blanchard 1850 se encuentra distribuido ampliamente en la región Neotropical y esta constituido por seis especies en Chile Sch. serratus Phil., Sch. breviventris Phil., Sch. longipes Phil., Sch. modestus Phil., Sch. ursulus Phil. y Sch. vestitus Phil.y dos en Brasil Sch. bicoloripes Blanch. y Sch. flavescens Blanch., según Blackwelder (1944). Recientes listados de Smith (2008a) y Mondaca (2008) señalan sólo cinco especies para Chile: Sch. serratus, Sch. breviventris, Sch. modestus, Sch. ursulus y Sch. vestitus. Smith (2008b) plantea un nuevo ordenamiento en la clasificación de algunos de estos insectos señalando a Sch. serratus y Sch. breviventris como sinonímias de Pristerophora picipennis (Solier) presente en Chile y Argentina. Los adultos de estos insectos son conocidos como “pololitos” y las larvas como “gusanos blancos” en todos sus estadios. La taxonomía de las larvas de estas especies es completamente desconocida. Las dos especies que se describen conforman una parte importante del ensamble de gusanos blancos en algunos ecosistemas pratenses en el sur de Chile. Ellos son consumidores de raíces de un amplio rango de especies pratenses y cultivos, causando severos daños a las plantas (Cisternas y Carrillo, 1989; Cisternas y Carrillo, 2001).

Tribu Lichniini Burmeister, 1844. A esta tribu pertenece la especie Arctodium mahdii. El género Arctodium Burmeister, 1844 se distribuye en la región Neotropical y esta constituido por cuatro especies, A. discolor Erichson, A. mahdii Hawkins, A. planum Blanchard y A. vulpinum Ericsson, (Hawkins, 2006; Evans y Smith, 2009). Estas especies se encuentran sólo en Chile desde la Region IV (Coquimbo) a la Region IX ( La Araucania). La morfología de las larvas de la tribu Lichniini es particularmente desconocida ya que no se ha descrito ninguna de sus especies, (Hawkins, 2006). Los adultos de esta tribu son polinizadores generalistas, llamados comúnmente “bumblebee scarabs” siendo característica su abundante pilosidad y sus larvas son llamadas “Gusanos blancos”. Las patas adaptadas para cavar de las hembras sugieren que la ovipostura ocurre

en el suelo. A. mahdii se distribuye entre la Region VII (Maule) a la Region IX (Araucania) (Hawkins, 2006).

Subfamilia Rutelinae MacLeay 1819. Los Rutelinae se dividen en las siguientes tribus Rutelini, Anatistini, Anoplognathini, Geniatini, Alvarengiini, Anomalini y Adoretini, (Smith, 2006).

Tribu Anoplognathini MacLeay 1819. Ha sido dividida en las siguientes subtribus: Anoplognathina, Schizognathina, Phalangogoniina, Platycoeliina y Brachysternina, (Smith, 2006).

Subtribu Brachysternina Burmeister, 1844. A esta subtribu pertenecen las especes H. elegans y A. punctatus. Solo cuatro especies de Anoplognatini en su estado larval han sido descritas previamente en el mundo, dos de ellas de Tasmania – Australia de los géneros Saulostomus y Anoplognathus (Hardy, 1976; McQuillan, 1985). Las otras especies son las Neotropicales del género Platycoelia (Paucar-Cabrera y Smith, 2002). Hylamorpha y Aulacopalpus son dos géneros endémicos pertenecientes a la Subtribu Brachysternina que se distribuyen en el cono sur de América (Chile y Argentina). H. elegans es la especie más común en los distintos agroecosistemas y de amplia distribución en Chile (Durán, 1952; Carrillo y Cerda, 1987; Ratcliffe y Ocampo, 2002). Los adultos son conocidos como “San Juanes”, “Pololos” o “Pololo verde chico”. A. punctatus es una especie circunscrita a las regiones del sur de Chile (Valdivia – Llanquihue), (Smith, 2002) y se desconoce su importancia relativa en los ensambles. Los adultos son conocidos como “San Juanes”, “Pololo café” o “Pololo de otoño”. Las larvas de estas especies de Rutelinae en todos sus estadios es conocida como gusano blanco, se desarrollan bajo el suelo, alimentándose de raíces de distintas plantas cultivadas y siendo parte de los ensambles de escarabaeidos en las praderas del sur de Chile. H. elegans es un insecto recurrente en los distintos ecosistemas, siendo el pratense el más regularmente atacado Aguilera et. al., (1996).

CUADRO 1. Posición sistemática de las especies de scararabaeidos que se describen en esta tesis de acuerdo a Evans y Smith (2009).

Orden Superfamilia Familia Subfamilia Tribu Subtribu Especie

Phytholaema herrmanni Germain

Incertae sedis Phytholaema dilutipes (Fairm. & Germ.) Pristerophora picipennis (Solier) Macrodactylini Schizochelus modestus Philippi

Melolonthinae Lichniini Arctodium mahdii Howkins Hylamorpha elegans (Burmeister) Coleoptera Rutelinae Anoplognathini Brachysternina Scarabaeidae Aulacopalpus punctatus (Fairm. & Germ.) Scarabaeoidea

Selección de sitio de ovipostura en adultos de escarabaeidos

La selección de un sitio adecuado de oviposición en los insectos fitófagos es un factor crucial en la sobrevivencia y posterior desarrollo de la progenie. Debido a que la distribución de las plantas es heterogénea, la selección de sitios adecuados es fundamental para el éxito de la descendencia (Thompson, 1988). Debido a la variación en el comportamiento de oviposición que se presenta en una misma especie (Thompson y Pellmyr, 1991), la selección natural debería favorecer lugares que fueran óptimos para la descendencia (Holland et al., 2004).

Esta situación es particularmente importante en insectos que se alimentan de un recurso limitado y en el cual, en sus estados inmaduros presenta un movimiento escaso (Suguira et al., 2007). Sin embargo la selección de sitios de oviposición también se da en insectos polífagos, en que el primer o primeros estadios larvales se ven limitados en su capacidad de búsqueda de recursos alimenticios, debido a su pequeño tamaño, como es el caso de algunas larvas del suelo (Szendrei e Isaacs, 2005)

Los escarabaeidos y otros insectos tienden a presentar distribuciones altamente agregadas según Andersen (1987) y Brown y Gange (1990), siendo la distribución espacial de un insecto una propiedad intrínseca de la especie, (Taylor, 1961) y resultado de la interacción intra e interespecífica y su hábitat (Hunter, 2001). Los estados preimaginales de escarabaeidos son afectados por las características físicas, químicas y ambientales del suelo, destacando la textura, compactación, pH, temperatura, humedad, materia orgánica y condición nutritiva del mismo (Laughlin, 1963; Ridsdill Smith, et al., 1975; Vittum y Tashiro, 1980; King et al., 1981; Regniere et al., 1981; Potter, 1983; Vittum, 1984; Curry, 1987; Villani y Wrigth, 1990) y probablemente por la selección de los sitios de oviposición por los adultos como ocurre en especies de otros ordenes (Gotthard et al., 2004).

Antecedentes de comportamiento de ovipostura y selección de sitios de oviposición de las especies nativas son escasos. Rivera, (1904) y Duran, (1954) señalan para P. herrmanni que el vuelo es crepuscular, a baja altura del suelo y ocurre entre las 18:30 y 20:00 horas. El adulto no ingiere alimento y las hembras grávidas buscan terrenos con vegetación para la oviposición. Duran, (1952) y Cisternas, (1986), señalan que H. elegans presenta un vuelo crepuscular y nocturno. En relación a la selección de sitios y tipo de vegetación, East y King, (1977) plantean que las especies pratenses, altura y densidad parecen tener poco o ningún efecto en la selección del sitio de oviposición en las especies Costelytra zelandica (White) y Heteronychus arador (Fabricius). Por otra parte Szendrei y Isaacs, (2005), desarrollaron bioensayos con Popilia japonica Newman para determinar las repuestas a varios factores derivados de las plantas que puedan influir en la selección del sitio de oviposición. Las hembras prefirieron poner sus huevos en tratamientos con ballicas que en pasto artificial y establecieron el rol de los objetos verticales en la superficie del suelo al comparar suelo desnudo con brotes artificiales. Las hembras fueron capaces de discriminar entre calidad y cantidad de brotes artificiales, la oviposición fue mayor donde los brotes fueron de mayor diámetro y más densos. Los resultados obtenidos por los autores sugieren que la oviposición de P. japonica es selectiva y esta influenciada por señales visuales y químicas derivadas de las plantas. Según Régnière et al., (1981) y Allsopp et al., (1992) en

experiencias de laboratorio las hembras fueron selectivas en su oviposición a características de suelo como textura, humedad y materia orgánica. Gaylor y Frankie (1979), establecieron que la humedad de suelo influye significativamente sobre la ovipostura de Phyllophaga crinita Burmeister, no hubo ovipostura en suelos extremadamente secos o extremadamente húmedos.

En esta investigación se estudiará el comportamiento de selección de sitio de oviposición de H. elegans y P. herrmanni por su importancia como plagas de cultivos y praderas, pero especialmente por el distinto comportamiento de oviposición que presentan ambas especies. Mientras H. elegans coloca sus huevos en los primeros 5 cm del suelo (Cisternas publicación en elaboración), donde esta más expuesta a factores como desecación, temperatura, etc, por lo cual la selección de un sitio de oviposición por la hembra es crucial para la embriogénesis del huevo (absorbe agua desde el suelo) (Fresard, 1992) y para el desarrollo de las larvas, por otro lado correspondería a una subfamilia cuyas larvas son capaces de alimentarse de la materia orgánica del suelo (Ritcher, 1958). P. herrmanni en cambio coloca sus huevos a mayor profundidad, lo cual le permite escapar al menos en parte a las variaciones en humedad y temperatura que pueden afectar la embriogénesis y el desarrollo larval, sin embargo las especies de la subfamilia Melolonthinae, en su estado larval, tendrían mayor necesidad de raíces de plantas para su normal desarrollo según Ritcher (1958).

Efecto del suelo y planta en el potencial biológico de los escarabaeidos

Factores bióticos como las enfermedades causadas por bacterias, hongos, protozoos, virus, nematodos y otros agentes (Kard et al., 1988; Villani y Wright, 1988; Glare y Jackson, 1992), además de factores abióticos como las características físicas y químicas del suelo afectan directamente la sobrevivencia, crecimiento y desarrollo de las larvas del suelo a través de factores como MO, pH, humedad y temperatura, estructura, porosidad y aireación, (Régniére et al.,1981; Potter y Gordon, 1984; Vitum, 1984; Curry, 1987; Brown y Gange,

1990, Mbata, 2004). La calidad del alimento esta determinada por las características físicas y químicas de las plantas las cuales influyen sobre la morfología, crecimiento, reproducción y sobrevivencia de los insectos criados durante el periodo larval, (Logan et al., 2001, Logan y Kettle, 2002; Karino et al., 2004). Larvas criadas bajo una nutrición pobre reducen la duración de su estado larval, pupan prematuramente y conducen a una emergencia temprana de adultos pequeños (Moczek, 1998; Shafiei et al., 2001). La presencia o ausencia de raíces en el suelo es un factor importante para el adecuado desarrollo de algunas especies, (Farrell 1972; Ridsdill Smith, 1975; Berry y Potter 1995; Logan y Kettle, 2002). Los exudados de las plantas no producen variaciones de peso en larvas de tercer estadio de H. elegans, (Rojas, 2005). La mayoría de los insectos que se alimentan de raíces son considerados herbívoros polífagos. Sin embargo, algunos exhiben preferencias por algunas especies, confirmando la existencia de especies favorables y desfavorables, (East y King, 1977; King et al., 1981; Prestidge et al.,1985). La adición de fertilizantes artificiales tuvo un pequeño a nulo efecto sobre el crecimiento y sobrevivencia de C. zealandica, (Prestidge et al., 1985). La alimentación de las larvas puede tener importantes implicacias en el desarrollo de las poblaciones de escarabaeidos más aun sobre aquellas especies que no se alimentan como adultos, donde el potencial de fecundidad esta relacionado a la alimentación larval, (Logan et al., 2001).

En experimentos sobre los efectos de factores abióticos y bióticos sobre el comportamiento de larvas de escarabaeidos se demostró, cómo altos niveles de humedad de suelo mitigan el efecto de la alimentación larval sobre las plantas (Ladd y Buriff, 1979).

King, et al., (1981), utilizando tres especies forrajeras para alimentar larvas de tercer estadio de H. arator, determinaron un mayor crecimiento de las larvas en aquellas alimentadas con gramíneas que con trébol blanco, debido a un menor consumo, probablemente por la presencia de compuestos antialimetarios en las raíces. Según Ridsdill Smith, (1975), muchas especies de escarabaeidos pueden alimentarse de MO y raíces vivas; pero la habilidad de selección de ellas es poco conocida. Carrillo et al. (2004) establecieron

que las larvas de P. herrmanni y H. elegans no responden a la presencia de plantas, lo que se debería a la capacidad de estas especies de alimentarse de MO del suelo. En experimentos realizados con S. nigrolineata, alimentados con dos tipos de suelos con y sin raíces se determinó que la tasa de crecimiento relativo fue mayor en los tratamientos con raíces vivas, aunque la larva pudo sobrevivir en ambos tipos de suelo en ausencia de raíces vivas (Ridsdill Smith, 1975).

Las larvas de varias especies de escarabaeidos crecen mejor en suelos con plantas vivas como A. austriaca, P. japonica, C. zelandica y S.geminata, siendo la excepción R. morbillosa (Ridsdill Smith, 1975).

Según Ritcher, (1958), el primer estadio larval de las especies que consumen raíces, se alimentan en parte de materia orgánica en el suelo y en el segundo y tercer estadio se alimentarían principalmente de raíces y vástagos bajo el suelo. De Fluiter citado por Ritcher, (1958), clasifica a las larvas de escarabaeidos según su hábito alimenticio:  Larvas que se alimentan sólo de materia orgánica muerta, (Cetoniinae).  Larvas que consumen materia orgánica; pero que en su ausencia consumen raíces, (Rutelinae y Dynastinae) y  Larvas que se alimentan principalmente de raíces de plantas, (Melolonthinae).

En base a lo anteriormente expuesto se estudiará el desarrollo de los estados inmaduros de H. elegans y P. herrmanni, en los principales suelos de la región sur, un suelo derivado de cenizas volcánicas recientes, un andisol (trumao), un suelo derivado de cenizas volcánicas mas antiguas, un ultisol (rojo arcilloso) y un suelo inceptisol (ñadi).

HIPÓTESIS

La selección del sitio de oviposición por H. elegans y P. herrmanni escarabaeidos nativos depende de estímulos captados por las hembras, producidas por factores abióticos y bióticos .

Las especies que conforman el ensamble de larvas de escarabaeidos pueden ser caracterizadas e identificadas a través de la taxonomía tradicional.

Las raíces de plantas vivas son necesarias para una óptima adecuación biológica (fitness) de las larvas de H. elegans y P. herrmanni.

El tipo de suelo afecta el desarrollo de las larvas de las especies H. elegans y P. herrmanni.

OBJETIVOS

Objetivos Generales

Estudiar en las especies H. elegans y P. herrmanni, componentes importantes del ensamble de larvas de escarabaeidos presentes en praderas, el rol del sustrato o suelo, su cobertura y la presencia de plantas y raíces vivas sobre la selección del sitio de oviposición y el desarrollo y crecimiento larval.

Describir las larvas de especies que forman parte del ensamble de escarabaeidos presentes en suelos con praderas en el sur de Chile.

Objetivos Específicos

Describir las estructuras de importancia taxonómica en larvas de escarabaeidos que permitan diferenciarlas a nivel de especie.

Determinar el efecto de la cobertura y tipo de sustrato o suelo en interacción con raíces y plantas herbáceas sobre la selección del sitio de oviposición en H. elegans y P. herrmanni.

Establecer el efecto de tres tipos de suelo de la Región de Los Lagos, Chile, con y sin presencia de raíces, sobre el desarrollo, crecimiento y mortalidad del estado larval de H. elegans y P. herrmanni.

2. DESCRIPCIÓN TAXONÓMICA DEL ESTADO LARVAL DE DOS ESPECIES DE SCARABAEIDAE: MELOLONTHINAE: TRIBU INSERTAE SEDIS.

Este artículo puede ser consultado bajo el título:

Cisternas, A. E. and R.LL. Carrillo. 2010. DESCRIPTION OF THE LARVAE OF Phytholaema herrmanni Germain. AND Phytholaema dilutipes (Fairmaire and Germain). (COLEOPTERA: SCARABAEIDAE: MELOLONTHINAE). The Colleopterist Bulletin 64 (2): 141-147.

DESCRIPTION OF THE LARVAE OF Phytholaema herrmanni GERMAIN AND Phytholaema dilutipes (FAIRMAIRE AND GERMAIN) (COLEOPTERA: SCARABAEIDAE: MELOLONTHINAE).

ERNESTO CISTERNAS A. Escuela de Graduados, Facultad de Ciencias Agrarias. Universidad Austral de Chile. Casilla 567, Valdivia, CHILE [email protected]

AND

ROBERTO CARRILLO LL. Instituto de Producción y Sanidad Vegetal, Facultad de Ciencias Agrarias. Universidad Austral de Chile. Casilla 567, Valdivia, CHILE [email protected]

ABSTRACT

The third instar of Phytholaema herrmanni Germain and Phytholaema dilutipes (Fairmaire and Germain) (Scarabaeidae: Melolonthinae) are described and illustrated. The larvae were reared under laboratory conditions from eggs laid by adults and larvae collected from pasture soil. This is the first description of larvae of the genus Phytholaema Blanchard. The larvae of both species have a vestigial sclerotized structure on the pleura of A9 and many other similar characteristics. The similar size of abdominal spiracles A6 and A8 in P. dilutipes is key to differentiating it from P. herrmanni, which has spiracle A8 smaller than spiracle A6.

Key Words: morphology, third instar, white grubs, spiracles

RESUMEN

Se describe e ilustra el tercer estadío de Phytholaema herrmanni Germain y Phytholaema dilutipes (Fairmaire y Germain) (Scarabaeidae: Melolonthinae). Las larvas fueron criadas bajo condiciones de laboratorio desde huevos puestos por los adultos y desde larvas colectadas desde el suelo bajo praderas. Esta es la primera descripción de larvas del género Phytholaema Blanchard. Las larvas de ambas especies presentan una estructura vestigial esclerotizada en la pleura de A9 y muchas otras características similares. El tamaño del espiráculo de A6 similar al espiráculo de A8 en P. dilutipes es clave para diferenciarla de P. herrmanni, la cual tiene el espiráculo de A8 más pequeño que el espiráculo A6.

INTRODUCTION

The lack of information about the morphological characteristics of the immature stages of Chilean scarabs has been a barrier to competent inter-guild and insect-plant relationship studies that can provide a substantial contribution to managing these insects as pests. Phytholaema Blanchard is found in the Neotropical region and includes three species, Phytholaema dilutipes (Fairmaire and Germain), Phytholaema herrmanni Germain, with two subspecies, P. herrmanni herrmanni Germain and P.herrmanni pallida Saylor, and Phytholaema mutabilis (Solier) (Evans and Smith 2009). All of these species are found in Chile; P. mutabilis is also found in Argentina. According to Evans and Smith (2009), the current tribal placement of Phytholaema is incertae sedis, thus more research is needed to properly classify this genus. The adults are known as “San Juanes”, “Pololos” or “Pololo café chico” and the larvae are called “white grubs”. The morphology of the larvae of these species is unknown. Despite this lack of this information, the larvae of Phytholaema are an important part of the scarab assemblage in pastures of the central and southern regions of Chile. Larvae attain high densities, feeding on the roots of a wide range of plants and causing considerable economic damage to cultivated plants (Duran 1954).

The objectives of our study were: 1) to describe the third instar of P. herrmanni and P. ditilupes and 2) to determine the specific morphological characters useful for distinguishing the two species.

MATERIAL AND METHODS

Third instars of P. herrmanni and P. dilutipes were collected from soil and reared until to adulthood to confirm their identification. Larvae were also obtained from laboratory-deposited eggs in plastic rearing boxes with sterilized soil. The larvae were reared in an acclimatized chamber at 20±2º C and 60±5%RH without a photoperiod. The rearing boxes were examined weekly. The larvae used for the morphological descriptions were fixed in a solution of KAAD (ethyl alcohol, kerosene, glacial acetic acid, and dioxane) (Carne 1951) and preserved in 70% alcohol. Morphological structures were mounted in Faure, Canada balsam, and Hoyer’s solutions. Drawings were made using a stereomicroscope, a dissecting microscope, a camera Lucida, and microphotography. Measurements were taken with an ocular micrometer. Voucher specimens were deposited in the Entomology Collection of the Universidad Austral de Chile, Valdivia.

Terminology from Ritcher (1966) was used in the descriptions of the larvae. The parts of the larvae for description have also been used by Hayes (1929), Peterson (1960), Edmonds and Halffter (1978), Vanin and Costa (1980), McQuillan (1985), Paucar-Cabrera and Smith (2002), Mico et al. (2003), and Grebennikov and Scholtz (2004).

RESULTS

Phytholaema herrmanni Germain, 1901 Third instar (Figs. 1-15)

The description is based on larvae reared from eggs laid by adults collected from pastures and meadows in Valdivia and the Santa Rosa Experimental Station of the Universidad Austral de Chile, in the Región de los Ríos, 39° 47’ S and 73º 14’ W at 13 m elevation.

Description. C-shaped. Head: width of cephalic capsule L1: 1.52±0.06 mm (n =

12); L2: 2.20 ± 0.11 mm (n = 19), and L3: 3.61±0.12 mm (n = 24). Ocellus absent. Cranium (Fig. 1): smooth, brilliant, light yellow; frontal suture whitish and bisinuate, forming a sharp angle at the joint; epicraneal suture ¼ of the length of the frontal suture; with 5-6 dorsoepicranial setae and 2 lateral setae on each side; anterior and posterior frontal setae present; 1 seta anterior adjacent to the antennal socket; 6-7 long setae adjacent and lateral to the antennal socket when viewed from the front. Antenna (Fig.2): four antennomeres; apical antennomere with 1 dorsal sensory spot and 2 ventral sensory spots; second antennomere 1.5× as long as first antennomere; third antennomere with an elongated process and a ventral sensory spot. Clypeus (Fig. 1): trapezoidal; surface of preclypeus smooth, lighter in color, without setae; postclypeus smooth with 2 long exterior setae on each side and 2 clypeal setae. Labrum (Fig.1): pentagonal, symmetrical, trilobed, apical lobe with a pair of blunt setae; 6-8 posterior setae, 4 medial setae, and 6 anterolateral setae present. Epipharynx (Fig. 3): pentagonal; with plegmatia, proplegmatium absent, epizygum sclerotized and closely connected to the right zygum, left zygum absent; corypha with 2 blunt setae; clithrum present; haptomerum with 10 – 12 sensilla and 3-4 heli in a transverse row (mode = 3); acanthoparia with 8-9 short, sickle-shaped setae; gymnoparia and chaetoparia setose; pternotorma, dexiotorma, and laeotorma well developed and sclerotized without epitorma or apotorma; laeotorma one-half length of dexiothorma;

haptolachus with 2 nesia; crepis present. Mandibles (Figs.4, 5): asymmetrical, left cutting region with 3 teeth and right with 2 teeth; scrobe with 7-8 setae and 7-8 basolateral setae; dorsal carina with 1 seta; dorso-molar area with a small group of short setae close to the molar; left ventral process smaller than right ventral process; calyx elongated; brustia present; acia well developed; stridulatory area absent; ventral processes roughened. Maxilla (Fig. 6): with galea and lacinia fused, but slightly separated distally; galea with well developed uncus; lacinia with 3 unci, the distal 2 fused at base (Fig. 7); stridulatory area formed by a row of 10-14 rounded teeth, some slightly overlapping (Fig. 8). Labium (Figs. 9, 10): hypopharyngeal sclerome asymmetrical, strongly developed and sclerotized, left hypopharyngeal lobe with a row of vertically oriented setae and proximal area of scleroma with two rows of horizontally oriented setae; glossa covered by slender, robust setae; subtrapezoidal postmentum with 2 slender setae; posterior section of prementum with 2 long setae and 2 short setae, anterior section of prementum with 8 slender setae and 3 lateral setae close to the labial palp.

Thorax. Spiracles (Fig.11): C-shaped respiratory plate, 0.30 mm long, 0.22 mm wide, with 9-12 irregular large holes across diameter (Fig.12); distance between the two lobes of respiratory plate 2/3 distance of diameter of dorso-ventral side of bulla. Pronotum: with lateral scalloped scleromas, yellow, irregular shape surrounded by 10–16 setae; with 3 irregular rows of long, medium-length, and short setae. Legs (Fig. 13): size of legs increases gradually from the prothorax to the metathorax; each tarsungulus affiliated with 2 proximal setae, tarsungulus of the metathoracic leg smaller.

Abdomen. Abdominal spiracles A1-4 of similar size (0.35-0.38 mm), A5 smaller (0.30 mm), A6-7 and T smaller (0.27-0.29 mm), A8 conspicuously smaller (0.19 mm); spiracular area with 1 very long seta and 4-6 short, medium-length, and long setae. Dorsum of A2–A6 with 3-4 rows of short setae. Pleural lobe A9 with a vestigial sclerotized structure (Fig. 14). Raster (Fig.15): with 2 parallel, longitudinal palidia, each with 9-16 pali; tegillum with 12- 22 hamate setae; septulum broad and bare; Y-shaped anal opening

covered with a mix of slender, straight and cylindrical medium-length and small setae; ventral anal lobes with slender, straight medium-length and small setae; campus with 2 moderately long setae; barbula not well defined, with a few slender setae.

Phytholaema dilutipes (Fairmaire and Germain, 1860) Third instar (Figs. 16-30)

The description is based on larvae collected from a pasture near Curepto, 7a Región del Maule 35º 2’ S and 72º 4’ W at 10 m elevation.

Description. C-shaped. Head: width of cephalic capsule L1: 1.43±0.09 mm (n =

4), L2: 2.05±0.18 mm (n = 4), and L3: 3.6±0.11 mm (n = 10); without ocellus. Cranium (Fig. 16): smooth, shiny, light yellow; frontal suture whitish, bisinuate, forming a sharp angle at the joint; epicranial suture 1/3 length of frontal suture; 4-5 dorso-epicraneal setae and 5 lateral setae on each side; 2 anterior setae and 3 frontal posterior setae; 1 seta anterior adjacent to antennal socket; 7-8 long setae adjacent lateral to antennal socket viewed from the front. Antenna (Fig.17): four antennomeres; apical antennomere with 1 dorsal sensory spot and 2 ventral sensory spots; second antennomere 1.5-1.8× longer than first antennomere; third antennomere with elongated process with 1 ventral sensory spot. Clypeus (Fig. 16): trapezoidal; surface of preclypeus smooth, without setae; smooth postclypeus with 2 long setae, 2 medium-length external setae and 2 clypeal setae. Labrum (Fig. 16): pentagonal, symmetrical, trilobed, apical lobe with a pair of blunt setae; with 6 – 10 posterior setae; 4 medial setae and 10 anterolateral setae present. Epipharynx (Fig. 18): pentagonal; plegmatia and proplegmatium absent; sclerotized epizygum closely connected to the right zigum, left zigum absent; corypha with 2 blunt setae; clithrum present; haptomerum with 9–11 sensilla and 3- 4 heli in a transverse row; acanthoparia with 8-10 sickle-shaped, short setae; gymnoparia without setae; haetoparia setose; dexiotorma and laeotorma well developed and sclerotized, without apotorma and epitorna; pternotorma present; laeotorma one-half length of dexiotorma; haptolachus with 2 nesia; crepis present.

Mandibles (Figs.19, 20): asymmetrical, left cutting region with 3 teeth and right with 2 teeth; scrobe with 7-8 setae and 2-3 basolateral setae; dorsal carina with 1 seta; dorso-molar area with a group of setae close to molar; left ventral process smaller than right ventral process; calyx elongated; brustia present; acia well developed; stridulatory area absent; ventral processes roughened. Maxilla (Fig. 21): galea and lacinia fused and slightly separated at the apex; galea with a well developed uncus; lacinia with 3 unci, the distal 2 fused at the base (Fig. 22); stridulatory area formed by a row of 9-11 rounded teeth (Fig. 23). Labium (Figs. 24- 25): hypopharyngeal sclerome asymmetrical, strongly developed and sclerotized, left hypopharyngeal lobe with a row of vertically oriented setae and proximal area of the scleroma with a row of horizontally oriented setae (Fig. 24); glossa covered by slender and robust setae; subtrapezoidal postmentum with 2 slender setae; posterior prementum with 2 long and 2 short setae, anterior prementum with 2 long setae and 2 short setae and 3-4 lateral setae close to the labial palp (Fig. 25).

Thorax. Spiracles (Fig. 26): C-shaped respiratory plate 0.30 mm long, 0.21 mm wide, with 12-15 irregular and small holes across diameter (Fig. 27); distance between the 2 lobes of the respiratory plate 2/3 diameter of dorso-ventral diameter of bulla. Pronotum: with an irregular, sclerotized shield, yellow on the side, surrounded by18-20 setae, 2-3 very long. Legs (Fig. 28): gradually increasing in size from prothoraxic to metathoracic leg; each tarsungulus affiliated with 2 proximal setae; tarsungulus of metathoraxic leg smaller.

Abdomen. Abdominal spiracles A1-4 of similar size (3.57-3.69 mm), A5, A7, and T smaller (2.76-2.95 mm), A6 and A8 conspicuously smaller (2.0-2.35 mm). Dorsum of abdominal segments A2–A6 with 3-6 rows of short setae. Pleural lobe A9 with vestigial sclerotized structure (Fig. 29). Spiracular area with one very long seta and 3-6 short, medium and long setae. Raster (Fig. 30): with 2 parallel, longitudinal palidia with 11-19 pali (n = 10); tegillum with 19- 28 hamate setae; septulum narrow and glabrous; anal opening Y-shaped; dorsal anal lobe covered with slender cylindrical setae, mastly short and medium-length; ventral anal lobe with slender cylindrical setae, mainly medium-length and

short; campus with 2 moderately long setae; barbula not well defined, with a few slender, long setae.

DISCUSSION

Distinct morphological differences between third instars of P. herrmanni and P. dilutipes are apparent. Both species have a Y-shaped anal opening, with two longitudinal, parallel palidia, but P. hermanni has 9-16 pali, whereas P. dilutipes has 11-19 pali. The tegillum of P. herrmanni has 12–22 hamate setae, but P. dilutipes has 19-28 hamate setae. The septulum is broad in P. herrmanni, but narrow in P. dilutipes. The spiracles on A6, A7, and T are similar in size in P. herrmanni; the spiracles on A6 are smaller than those on A7 and T in P. dilutipes. The respiratory plate of the thoracic spiracle of P. herrmanni has 9-12 irregular and large holes across the diameter, whereas in P. diutipes, the respiratory plate of the thoracic spiracle has 12-15 irregular and small holes across diameter. The maxillary stridulatory area of P. herrmanni is formed by a row of 10-14 rounded teeth, but the maxillary stridulatory area of P. dilutipes is formed by a row of 9-11 rounded teeth.

ACKNOWLEDGMENTS

We thank Mario Elgueta, National Museum of Natural History (MNHN), Santiago, Chile for identifying the adults. We also thank Gustavo Valdebenito, who collaborated with us in the field collections and Leticia Silvestre for her collaboration in the laboratory work.

REFERENCES CITED

Carne, P. B. 1951. Preservation techniques for scarabaeid and other insect larvae. Proceedings of the Linnean Society of New South Wales 76: 26-30.

Durán, L. 1954. La biología de Phytholaema herrmanni Germ.y mención de otros escarabaeido perjudiciales a la agricultura de las provincias australes de Chile. Revista Chilena de Historia Natural 54: 5-50. Edmonds, W. D., and G. Halffter. 1978. Taxonomic review of immature dung beetles of the subfamily Scarabaeinae (Coleoptera: Scarabaeidae). Systematic Entomology 3: 307- 331. Evans, A. V., and A. B. T. Smith. 2009. An electronic checklist of the New World chafers (Coleoptera: Scarabaeidae: Melolonthinae) Version 3. Available at: www- museum.unl.edu/research/entomology/SSSA/nwmelos.htm (Accessed on 01/29/2010). Grebennikov, V. V., and C. H. Scholtz. 2004. The basal phylogeny of Scarabaeoidea (Insecta: Coleoptera) inferred from larval morphology. Invertebrate Systematics 18: 321- 348. Hayes, W. P. 1929. Morphology, taxonomy, and biology of larval Scarabaeoidea. Illinois Biological Monographs 12(2): 1-125. McQuillan, P. B. 1985. The identification of root-feeding cockchafer larvae (Coleoptera: Scarabaeidae) found in pastures in Tasmania. Australian Journal of Zoology 33: 509-546. Micó, E., M. A. Morón, and E. Galante. 2003. New larval description and biology of some New World Anomalini beetles (Scarabaeidae: Rutelinae). Annals of the Entomological Society of America 96(5): 597-614. Paucar-Cabrera, A., and A. B. T. Smith. 2002. Larval descriptions for the Neotropical genus Platycoelia (Coleoptera: Scarabaeidae: Rutelinae: Anoplognathini). The Coleopterist Bulletin 56(3): 438-445. Peterson, A. 1960. Larvae of Insects. An Introduction to Neartic species. Part II. Coleoptera, Diptera, Neuroptera, Siphonaptera, Mecoptera, Trichoptera. Privately published, Columbus, OH. 218 pp. Ritcher, P. 1966. White Grubs and their Allies. A Study of North American Scarabaeoidea Larvae. University of Oregon Press, Corvallis, OR. 219 pp. Vanin, S. A., and C. Costa. 1980. Larvae of Neotropical Coleoptera III: Scarabaeidae, Rutelinae. Papéis Avulsos de Zoología 33: 275-283.

0,2 1.0 1 3

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Figs. 1-8. Phytholaema herrmanni, third instar. 1) Head, frontal view, distal antennomere dorsal view; 2) Distal antennomere lateral view; 3) Epipharynx; 4) Mandibles, ventral view a) left and b) right; 5) Mandibles, dorsal view a) right and b) left; 6) Maxilla, a) dorsal view and b) ventral view; 7) Apex of lacinia and galea showing unci; 8) Detail of maxillary stridulatory area. Bar = mm.

Figs. 9-15. Phytholaema herrmanni, third instar. 9) Labium dorsal view; 10) Hypopharynx; 11) Thoracic spiracle; 12) Respiratory plate; 13) Tarsungulus of a) prothoracic leg, b) mesothoracic leg, and c) metathoracic leg; 14) Sclerotized vestigial structure on A9; 15) Last abdominal segment, ventral view of raster. Bar = mm.

Figs. 16-23. Phytholaema dilutipes, third instar. 16) Head, frontal view, distal antennomere dorsal view; 17) Distal antennomere, a) ventral view, b) lateral view; 18) Epipharynx; 19) Mandibles, ventral view a) left and b) right; 20) Mandibles, dorsal view a) right and b) left; 21) Maxilla, a) dorsal and b) ventral view; 22) Apex of lacinia and galea showing unci; 23) Detail of maxillary stridulatory area. Bar = mm.

Figs. 24-30. Phytholaema dilutipes, third instar. 24) Labium, dorsal view; 25) Hypopharynx; 26) Thoracic spiracle; 27) Respiratory plate; 28) Tarsungulus of a) prothoracic leg, b) mesothoracic leg, and c) metathoracic leg; 29) Sclerotized vestigial structure on A9; 30) Last abdominal segment, ventral view of raster. Bar = mm.

3. DESCRIPCIÓN TAXONÓMICA DEL ESTADO LARVAL DE DOS ESPECIES DE SCARABAEIDAE: MELOLONTHINAE: MACRODACTYLINI

Este artículo puede ser consultado bajo el título:

Cisternas, A. E. y Carrillo, Ll. R. DESCRIPTION OF THE THIRD INSTARS OF Pristerophora picipennis (Solier) AND Schizochelus modestus Philippi FROM SOURTHERN CHILE (COLEOPTERA: SCARABAEIDAE: MELOLONTHINAE: MACRODACTYLINI). Accepted in The Colleopterist Bulletin (N° 1284)

DESCRIPTION OF THE THIRD INSTAR OF Pristerophora picipennis (Solier) AND Schizochelus modestus Philippi FROM SOUTHERN CHILE (COLEOPTERA: SCARABAEIDAE: MELOLONTHINAE: MACRODACTYLINI)

ERNESTO CISTERNAS A. Escuela de Graduados, Facultad de Ciencias Agrarias. Universidad Austral de Chile. Casilla 567, Valdivia, CHILE [email protected] AND ROBERTO CARRILLO LL. Instituto de Producción y Sanidad Vegetal, Facultad de Ciencias Agrarias. Universidad Austral de Chile. Casilla 567, Valdivia, CHILE [email protected]

ABSTRACT The third instar of Pristerophora picipennis (Solier) and Schizochelus modestus Philippi (Scarabaeidae: Melolonthinae: Macrodactylini) are described and illustrated. The descriptions are based on larvae reared from eggs laid by adults in the laboratory and on larvae collected in the field. These are the first descriptions of Macrodactylini larvae that live and feed in the root zone of pastures in southern Chile. The larvae of the two species can be distinguished easily by the presence or absence of palidia and the shape of the seta that cover the dorsum and the dorsal anal lobe. Setae on the dorsum and the dorsal anal lobe are sickle-shaped in S. modestus.

Key Words: white grubs, pasture, Chile, third instar, shape setae

RESUMEN

Se describe e ilustra las características del tercer estadio de Pristerophora picipennis (Solier) and Schizochelus modestus Philippi (Scarabaeidae: Melolonthinae: Macrodactylini). Las descripciones se basan en larvas criadas desde huevos puestos por adultos en el laboratorio y en larvas colectadas del suelo bajo praderas. Estas son las primeras descripciones de larvas de Macrodactylini que viven y se alimentan de la zona radicular en las praderas del sur de Chile. Las larvas de ambas especies pueden ser fácilmente separadas por la presencia o ausencia de palidia y la forma de las setas que cubren el dorso y el lóbulo anal dorsal. En S. modestus las setas del dorso y lóbulo anal dorsal presentan forma de hoz.

INTRODUCTION

The genus Schizochelus Blanchard is widely distributed in the Neotropical region and, according to Blackwelder (1944), is represented in Chile by six species: Schizochelus serratus Philippi, Schizochelus breviventris Philippi, Schizochelus longipes Philippi, Schizochelus modestus Philippi, Schizochelus ursulus Philippi, and Schizochelus vestitus Philippi. Two species, Schizochelus bicoloripes Blanchard and Schizochelus flavescens Blanchard, occur in Brazil. Recent listings by Evans and Smith (2007) and Mondaca (2008) indicate only five species for Chile: S. serratus, S. breviventris, S. modestus, S. ursulus, and S. vestitus. Smith (2008) and Evans and Smith (2009) proposed a new ordering in the classification of some of these insects, considering S. serratus and S. breviventris as synonymous to Pristerophora picipennis (Solier), which is present in Chile and Argentina. The adults of these insects are known as “pololitos” and the larvae are called “white grubs”. The two species whose larvae are described herein form an important part of the white grub assemblage in some pasture ecosystems in southern Chile. They are important consumers of the roots of a wide range of pasture species and crops, causing significant damage to plants (Cisternas and Carrillo 1989, 2001).

The morphological characteristics of the immature stages of Chilean Scarabaeidae has hardly been studied, albeit a fundamental base for biological, ecological, and behavioral knowledge, as well as for establishing their relative importance in the white grub assemblage and their insect-plant relations. The objectives of this study were 1) to describe the third instar of P. picipennis and S. modestus and 2) to determine the specific morphological characters to distinguish the two species.

MATERIAL AND METHODS

The third instars used for the descriptions of the two species were obtained through laboratory rearing of eggs laid in the laboratory, as well as from larvae collected in the field for rearing to adulthood. The insects were reared individually in plastic boxes with sieved soil and maintained in an acclimatized chamber at 20 ± 2º C, 60±5% RH, and no photoperiod. The rearing boxes were examined weekly, with replacement of moisture lost over the intervening period and removal of dead insects. The larvae used for morphological description were fixed in a solution of KAAD Mixture (ethyl alcohol, kerosene, glacial acetic, and dioxane) (Carne 1951) and preserved in 70% alcohol. Morphological structures were mounted in Faure, Canada balsam and Hoyer solutions. Drawings were made using a stereomicroscope, a microscope, camera Lucida, and microphotography. Measurements were taken with an ocular micrometer. Voucher specimens were deposited in the Entomology Collection of the Universidad Austral de Chile, Valdivia. The terminology of Ritcher (1966) was used in the description of the larvae. The parts of the larvae for description have also been used by Hayes (1929), Peterson (1960), Edmonds and Halffter (1978), Vanin and Costa (1980), McQuillan (1985), and Grebennikov and Scholtz (2004).

RESULTS

Pristerophora picipennis (Solier) Third Instar (Figs. 1-14)

The description is based on larvae reared from eggs laid by adults that were collected in flight in pastures near Puerto Varas 41° 23´S 73° 00´W, at 91 m elevation, and on larvae collected from pasture soil near Osorno, Region de Los Lagos 40° 31´S 73° 03´W, at 77 m elevation.

Description. C-shaped larvae. Head capsule: Width of the head capsule L1:

0.82±0.01 mm (n=6); L2: 1.27±0.05mm (n=15) and L3: 2.01±0.08 mm (n = 34). Ocellus absent. Cranium (Fig. 1): smooth, brilliant, light yellow; frontal suture whitish and bisinuate, forming a sharp angle at the joint; epicranial suture 1/3 of the length of the frontal suture; 3-6 dorsoepicranial setae; 2 frontal, posterior and anterior setae absent; 1 seta adjacent to the antennal socket; 4 long setae adjacent and lateral to the antennal socket when viewed from the front. Antenna (Fig.2): four antennomeres. Apical antennomere with 1 dorsal sensory spot and 2 ventral sensory spots. Second antennomere is 1.6 times as long as the first antennomere. Third antennomere with an elongate process and a ventral sensory spot. Clypeus (Fig. 1): trapezoidal shape. Surface of the preclypeus is smooth, lighter in color, without setae. Postclypeus smooth with 2 long exterior setae on each side and 2 clypeal setae. Labrum (Fig.1): pentagonal shape, symmetrical, trilobular, rugose, apical lobule with a pair of blunt setae; with 2 posterior setae, 4 medium-sized setae and 2 setae in the anterior margin. Epipharynx (Fig. 3): pentagonal shape, with plegmatia and proplegmatium absent, sclerosed epizygum closely connected to the right zigum, left zigum absent. Corypha with 2 blunt setae. Clithrum present. Acroparia with long and slender setae. Haptomerum with 10 – 12 sensilla and 3- 5 heli (mode: 4) in a transversal row. Acanthoparia with 2 thick apical setae and 7-9 sickle-shaped short setae by the side. Gymnoparia without setae. Chaetoparia with setae. Pternotorma, dexiotorma and laeotorma

well developed and sclerosed, without apotorma and epitorma. Laeotorma similar in length to the dexiothorma. Haptolachus with 2 nesium. Crepis absent. Mandibles (Figs.4a, 4b, 5a, 5b): asymmetrical. Left cutting region with 3 teeth and right with 2 teeth. Scrobe with 4 basolateral setae. Dorsal carina with 1 seta. Dorso-molar area with a small group of short setae close to the molar surface. Right ventral process is smaller than the left ventral process. Calx elongated. Brustia present. Acia well developed. Stridulatory area absent. Ventral processes with roughness. Maxilla (Fig. 6a - 6b): with the galea and lacinia fused and slightly separated at the apex; galea with a well developed uncus; lacinia with 2 unci (Fig. 7). Maxillary stridulatory area formed by a row of 8-12 teeth (Fig. 8). Labium (Figs. 9 - 10): hypopharygeal sclerome asymmetrical, strongly developed and sclerosed, left hypopharyngeal lobule with a row of vertically oriented setae and proximal area of the scleroma with a row of horizontally oriented setae. Glossa is covered by slender and robust setae. Sub-trapezoidal postmentum with 2 slender and long setae. Posterior prementum with 2 setae, anterior prementum with 8 slender setae and 1 short lateral setae close to the labial palp.

Thorax. Spiracles (Fig.11): C-shaped respiratory plate 0.13 mm long, 0.10 mm wide, with 9-12 irregular holes across diameter (Fig 12). The distance between the 2 lobules of the respiratory plate is equal to the diameter of the dorsoventral diameter of the bulla. Pronotum with sclerosed shield, yellow on each side, surrounded 15 -17 setae. Legs (Fig. 13 a,b,c): gradually increase in size from the prothoracic leg to the metathoracic. Each tarsunguli affiliated with 2 proximal setae. Tarsunguli of the metathoracic leg is smaller.

Abdomen. Abdominal spiracles A1-A2-A3-A4 and T of similar size, A5-A6 and A7 of the same size and smaller than the preceding ones, A8 conspicuously smaller. Spiraclular area with 3-10 short, medium and long setae. Dorso of the abdominal segments A1–A6 with 3 to 4 rows of short, sharp setae and one row with slender and long setae.

Raster (Fig.15): without palidia. Tegillum with 21-30 hamate setae. Y-shaped anal opening. Dorsal anal lobule densely covered with short, slender, straight and sharp setae. Ventral anal lobule with short, slender, straight, and sharp setae. Campus with 2 moderately long setae and 2 lateral slender setae. Barbula with slender setae.

Diagnosis. The larva of P. picipennis has a Y-shaped anal opening, without palidia. Tegillum with 21-30 hamate setae. Spiracles A1-A2-A3-A4 and T of similar size, A5-A6 and A7 of the same size and smaller than the previous ones, A8 conspicuously smaller. Apical antennomere with 1 dorsal sensory spot and 2 ventral sensory spots. Respiratory plate of the thoracic spiracle with 9-12 irregular holes across diameter. Maxillary stridulatory area formed by a row of 8-12 teeth. Dorso of the abdominal segments and dorsal anal lobule covered with straight slender, pointed and short setae.

Schizochelus modestus Philippi Third Instar (Figs. 15-28)

The larval description is based on the third instar larvae, reared from eggs laid by adults collected in flight in pastures near Puerto Varas 41° 23´S 73°00´W, at 91 m elevation, and on larvae from locations including Osorno, Río Bueno, Ranco, and Puyehue, all located in the Región de Los Lagos and Región de Los Ríos.

Description. C-shaped larvae. Head capsule: Width of the head capsule L3 1.67±0.078 mm (n = 10). Without ocellus. Cranium (Fig. 15): smooth, shiny, light yellow; whitish frontal suture, bisinuate, forming a sharp angle at the joint; epicranial suture 1/3 of the length of the frontal suture; 2 dorsoepicranial setae ; 2 frontal anterior setae and 2 frontal posterior setae; 1 seta adjacent to the antennal socket; 6-7 long setae adjacent to the lateral antennal socket viewed from the front. Antenna (Fig.16): With four antennomeres. Apical antennomere with 1 dorsal sensory spot and 2 ventral sensory spots. Second antennomere 1.8 times as long as the first antennomere. Third antennomere with elongated process with 1 ventral sensory spot. Clypeus (Fig.15): trapezoidal shape. Surface of the

preclypeus smooth, without setae. Smooth postclypeus with 2 lateral setae, 1 very long and 1 medium-length setae and 2 clypeal setae. Labrum (Fig.15): pentagonal shape, symmetrical, trilobular, apical lobule with a pair of blunt setae; with 2 posterior setae; 4 medial (position) setae and 2 setae in the anterior margin. Epipharynx (Fig.17): pentagonal shape, with plegmatia and proplegmatium absent, epizygum and zigum absent. Corypha with 2 blunt setae. Clithrum present. Haptomerum with 5–7 sensillum and 3- 4 heli in a transversal row. Acroparia with long thick setae. Acanthoparia with 5-7 sickle-shaped short posterior setae and 3 straight anterior setae. Gymnoparia without setae. Chaetoparia covered with setae. Pterotorma dexiotorma and laeotorma well developed and sclerosed without apotorma and epitorna. Dexiotorma 1,25 the length of the laeotorma. Haptolachus with 2 nesium. Crepis absent. Mandibles (Figs. 18a, b, 19a, b): asymmetrical. Left cutting region with 3 teeth and right with 2 teeth. Scrobe with 4-5 basolateral setae. Dorsal carina without seta. Dorso-molar area with a group of short setae close to the molar surface. Right ventral process is smaller than the left ventral process. Calx elongated. Brustia present. Acia well developed. Stridulatory area absent. Ventral processes with roughness. Maxilla (Fig. 20a, 20b): with the galea and lacinia fused and slightly separated at the apex; galea with a well developed uncus ; lacinia with 3 unci, the 2 distals are fused at the base (Fig. 21). Maxillary stridulatory area formed by a row of 8-10 teeth (Fig. 22). Labium (Figs. 23- 24): hypopharineal sclerome asymmetrical, strongly developed and sclerosed, left hypopharyngeal lobule with a row of horizontally oriented setae and proximal area of the scleroma with a row of vertically and horizontally oriented thick setae (Fig. 23). Glossa is covered by slender setae and robust setae. Sub-trapezoidal postmentum with 2 slender setae. Posterior prementum with 2 long setae, anterior prementum with 6 slender setae and 1 lateral setae close to the labial palp (Fig. 24).

Thorax. Spiracles (Fig.25): C-shaped respiratory plate 0.12 mm long, 0.05 mm wide, with 10-12 regular holes across diameter (Fig 26). The distance between the 2 lobules of the respiratory plate is similar to the dorsoventral diameter of the bulla. Pronotum with an irregular sclerosed shield, yellow on the side, surrounded by 15 -20 long, medium and short

setae. Dorsum of prothorax with 1 irregular row of long, medium-length and short setas. Legs (Fig. 27a-c): gradually increase in size from the prothoracic to the metathoracic legs. Each tarsunguli affiliated with 2 proximal setae. Tarsunguli of the metathoracic leg is smaller.

Abdomen. Abdominal spiracles A1 (0.09 mm), A2-A8 of a similar size (0.07 mm). Spiraclular area with one very long seta 4-6 short, medium and long setae. Dorso of the abdominal segments A1–A6 with 3 to 4 rows of short sickle setae. Raster (Fig.28): with 2 parallel longitudinal palidia with 9-10 pali (n=10). Tegillum with 13- 16 hamate setae by the side. Septulum bare. Y-shaped anal opening. Dorsal anal lobule covered with sickle- shaped setae mixed with straight thin small setae. Ventral anal lobule with straight and slender setae. Campus with 2 moderately long setae. Barbula with a few long setae.

Diagnosis. The larva of S. modestus has a Y-shaped anal opening. Raster with 2 parallel longitudinal palidium with 9-10 pali. Tegillum with 13- 16 hamate setae. Spiracles A1 (0,09 mm) and A2-A8 of a similar size (0.07 mm). Respiratory plate of the thoracic spiracle with 10-12 regular holes across diameter. Apical antennomere with 1 dorsal sensory spot and 2 ventral sensory spots. Maxillary stridulatory area formed by a row of 8- 10 teeth. Dorsum of the abdominal segments A1–A6 with 3 to 4 rows of short sickle setae. Dorsal anal lobule covered with sickle-shaped setae

ACKNOWLEDGMENTS

We are grateful to Mario Elgueta D. of the National Museum of Natural History, Santiago, Chile and Andrew B. T. Smith of the Canadian Museum of Nature, Ottawa, Canada, for taxonomic support in the identification of the adult Scarabaeidae. We are also grateful for the assistance of Gustavo Valdebenito, field assistant, and Leticia Silvestre for her collaboration in the laboratory work.

REFERENCES CITED

Blackwelder, R.E. 1944. Checklist of the coleopterous insect of Mexico, Central América, the West Indies and South America. United States Nacional Museum Bulletin 185(2): 189-265. Carne, P.B. 1951. Preservation techniques for scarabaeid and other insects larvae. Proceeding of the Linnean Society of New South Wales 76: 26-30. Cisternas, E. and R, Carrillo. 1989. Ciclo estacional de Schizochelus serratus Phil. (Coleoptera: Scarabaeidae). Revista Chilena de Entomología. 17: 61-63 Cisternas, E. and R. Carrillo. 2001. Seasonal soil vertical distribution of Schizochelus serratus Phil. (Coleoptera: Scarabaeidae). Revista Chilena de Entomología. 28: 95-98. Edmonds, W.D., and G. Halffter. 1978. Taxonomic review of immature dung beetles of the subfamily Scarabaeidae (Coleoptera: Scarabaeidae). Systematic Entomology. 3: 307- 331. Evans, A.V. and Smith, A. B. T. 2007. An electronic checklist of the new world chafers (Coleoptera : Sacrabaeidae: Melolonthinae) Version 2. Available from: http:/www.museum.unl.edu/research/ entomology/Guide/Scarabaeoidea/Scarabaidae/ Melolonthinae/Melolonthinae-Catalog/Pachydmini.pdf. (Accessed on 15 August 2008). Evans, A.V. and Smith, A. B. T. 2009. An electronic checklist of the new world chafers (Coleoptera: Scarabaeidae: Melolonthinae) Version 3. Electronically published, Otawa, Canada. 353 pp.Available from: http://www-museum.unl.edu/research/entomology /SSSA /nwmelos.htm Grebennikov, V.V., and Scholtz, C. H. 2004. The basal phylogeny of scarabaeoidea ((Insecta : Coleoptera) inferred from larval morphology. Invertebrate Systematics 18: 321- 348 Hayes, W. P. 1929. Morphology, taxonomy and biology of larval Scarabaeoidea. Illinois, Biological Monographs 12(2): 1-125.

McQuillan, P. B. 1985. The identification of root feeding cockchafer lavae (Coleptera: Scarabaeidae) found in pastures in Tasmania Australian Journal of Zoology 33: 509-546. Mondaca, J. E. 2008. Listado preliminar de los Scarabaeoidea (Coleoptera) de Chile. Available from: http://www.insectos.cl/listas/index_scarabaeoidea.php. (accessed on 31/09/2008). Peterson, A. 1960. Larvae of insects. An introduction to Neartic Species. Part II. Coleoptera, Diptera, Neuroptera, Siphonaptera, Mecoptera, Trichoptera. Privately published, Columbus, Ohio. 218 p. Ritcher, P. 1966. White grubs and their allies. A study of North American Scarabaeoida larvae. Corvallis, Oregon. University of Oregon Press, 219 p. Smith, A. B. 2008. South American Melolonthinae (Coleoptera: Scarabaeidae) classification and nomenclature: some problems and solutions. Insecta Mundi 60: 1-28. Vanin, S. A., and C. Costa. 1980. Larvae of Neotropical Coleoptera III: Scarabaeidae, Rutelinae. Papéis Avulsos de Zoología. 33(17): 275-283.

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7 8 5a 5b

Figs. 1-8. Pristerophora picipennis, third instar. 1) Head, frontal view, distal antennomere dorsal view; 2) Distal antennomere lateral view; 3) Epipharynx; 4) Mandibles, ventral view a) left, b) right; 5) Mandibles, dorsal view a) right, b) left; 6) Maxilla a) dorsal view, b) ventral view; 7) Apex of lacinia and galea showing unci; 8) Detail of maxillary stridulatory area. Bar = mm.

0,5 0,5 0,2 9 10 11 0,1

13a 0,01 13b 13c 0,2 12

1.0 Figs. 9-14. Pristerophora picipennis, third instar. 9) Labium dorsal view; 10) Hypopharynx; 11) Thoracic spiracle; 12) Respiratory plate; 13) Tarsungulus of a) prothoracic leg; b) mesothoracic leg; c) metathoracic leg 14) Last abdominal segment, ventral view with raster. Bar = mm.

0.5 c 17

15 1.0 16 0.2

20a 0.2 20b

18a 18b 0.5

0.1 0.2 19a 19b 21 22

Figs. 15- 22. Schizochelus modestus, third instar. 15) Head, frontal view, 16) Distal antennomere ,a) dorsal ; b) ventral ;and c) lateral view; 17) Epipharynx; 18) Mandibles, ventral view, a) left and b) right; 19) Mandibles, dorsal view, a) right and b) left; 20) Maxillae, a) dorsal view and b) ventral view; 21) Apex of lacinia and galea showing unci; 22) Maxillary stridulatory area. Bar = mm.

0,1 0,2 0,2 23 24 25

0,5 27a 27b 27c 0,05 26

1.0

Figs. 23-28. Schizochelus modestus, third instar. 23) Labium dorsal view; 24) Hypopharynx; 25) Thoracic spiracle; 26) Respiratory plate; 27) Tarsungulus of a) prothoracic leg, b) mesothoracic leg, c) metathoracic leg; 28) Last abdominal segment, ventral view of raster. Bar = mm.

4. DESCRIPCIÓN TAXONÓMICA DEL ESTADO LARVAL DE UNA ESPECIE DE SCARABAEIDAE: MELOLONTHINAE: LICHNIINI

Este artículo puede ser consultado bajo el título:

Cisternas, A. E. y Carrillo, Ll. R. 2010. LARVAL TAXONOMY OF Arctodium mahdii Hawkins LICHNIINI, ENDEMIC TO SOUTHERN CHILE (COLEOPTERA: SCARABAEIDAE: MELOLONTHINAE: LICHNIINI). The Colleopterist Bulletin 64 (4): 359-363.

DESCRIPTION OF THE THIRD INSTAR OF Arctodium mahdii Hawkins (COLEOPTERA: SCARABAEIDAE: MELOLONTHINAE: LICHNIINI)

ABSTRACT

A description and illustrations of the third instar of Arctodium mahdii Hawkins (Coleoptera: Scarabaeidae: Melolonthinae: Lichniini), endemic to southern Chile are given. Larvae were reared from eggs laid by adults under laboratory conditions and from larvae collected from pasture soil. Larvae of this species feed and live in the root zones of plant species present in southern Chilean pastures. Characteristics of this species are its separated galea and lacinia, very curved maxillary palpus, size and orientation of abdominal spiracles, Y-shaped anal opening, absence of palidia, and the distribution and shape of setae on the raster.

RESUMEN Se describe e ilustra el tercer estadio larval de Arctodium mahdii Hawkins, (Coleoptera: Scarabaeidae: Melolonthinae: Lichniini), endémico de la zona sur de Chile. Las larvas fueron obtenidas a través de la crianza bajo condiciones de laboratorio desde

huevos puestos por adultos y desde larvas colectadas desde el suelo bajo praderas. Las larvas de esta especie se alimentan y viven en la zona radical de las especies presentes en las praderas en el sur de Chile. Característico de la especie es su galea y lacinia separadas, palpo maxilar muy curvado, tamaño y orientación de los espiráculos abdominales, abertura anal en Y, ausencia de palidia y distribución y forma de setas en el ráster.

INTRODUCTION

The morphological characteristics of the immature stages of Chilean Scarabaeidae have hardly been studied. This knowledge is fundamental to understand insect-plant relationships, to establish the relative importance of these beetles, and to know the behavior, ecology, biology and ontogeny of the species.

The genus Arctodium Burmeister, 1844 is distributed in the Neotropical region and is represented by four species: A. discolor Erichson, A. mahdii Hawkins, A. planum Blanchard and A. vulpinum Erichson (Hawkins 2006; Evans and Smith 2009). These species are found only in Chile from the Región de Coquimbo to the Región de La Araucania. The morphology of the larvae of the tribe Lichniini is unknown, given that none of the larvae have been described (Hawkins 2006). The adults of this tribe are generalist pollinators, commonly called “bumblebee scarabs” because of their characteristic abundant pilosity. Arctodium mahdii is found from the Región del Maule to the Región de la Araucania (Hawkins 2006). However, the distribution of the species is broadened to the Región de Los Ríos with the collection of specimens for this description. The larvae are not commonly found, and when they are, their participation in the pasture assemblage is usually low. These larvae have also been found in the roots zone, forming part of Scarabaeidae assemblage associated with crops of strawberry and blueberry in the Región de la Araucania and Región de Los Ríos.

The objectives of our study were 1) to describe the third larval stage of A. mahdii and 2) to determine the specific morphological characters to distinguish the specie.

MATERIAL AND METHODS

The larval material used for the description was obtained through the laboratory rearing of adults to obtain eggs and the different larval stages, as well as larvae collected in the field for rearing to adulthood. The beetles were reared individually in plastic boxes with sieved soil and maintained in an acclimatized chamber at 20 ± 2ºC, 60±5 % RH without a photoperiod (L:D 0:24). The rearing boxes were examined weekly to replace lost moisture and remove dead insects.

The larvae used for the description were fixed in a solution of KAAD mixture (ethyl alcohol, kerosene, glacial acetic and dioxane) (Carne 1951) and preserved in 70% alcohol. The different morphological structures were mounted in Faure, Canada balsam or Hoyer solutions. The drawings were made using a stereomicroscope, a microscope, camera lucida and microphotography. Measurements were taken with an ocular micrometer. Voucher specimens were deposited in the Entomology Collection of the Universidad Austral de Chile, Valdivia. The terminology of Ritcher (1966) was used in the description of the larvae. The parts of the larvae for description have also been used by Hayes (1929), Peterson (1960), Edmonds and Halffter (1978), McQuillan (1985) and Grebennikov and Scholtz (2004).

RESULTS

Arctodium mahdii Hawkins, 2006 Third instar (Figs. 1-15)

The larval description is based on the third instars reared from eggs laid by adults and larvae collected from pasture soils near Lanco, Región de Los Rios, S 39° 29´ and W 72° 34´, 204 m elevation.

Description.C-shaped. Head : width of head capsule L1: 0.85±0.01 mm (n=6), L2:

1.35±0.04 mm (n=5) and L3: 2.25±0.03 mm (n = 10). Ocellus absent. Cranium (Fig. 1): smooth, shining, light yellow; frontal suture whitish and bisinuate, forming a sharp angle; epicraneal suture 1/3 length of frontal suture; 5 dorsoepicraneal setae, 10-12 posterior frontal setae and 10-12 anterior frontal setae; 2 setae adjacent to antennal socket; 10-12 long, medium-sized and small setae adjacent and lateral to antennal socket when viewed from the front. Antenna (Fig.2): with four antennomeres. Apical antennomere with 1 dorsal sensory spot and 2 ventral sensory spots. Second antennomere 1.1 times as long as first antennomere. Third antennomere with elongated process and a ventral sensory spot. Clypeus (Fig. 1): trapezoidal; surface of preclypeus smooth, lighter in color, without setae. Postclypeus smooth, with 3 exterior setae on each side and 8-10 clypeal setae. Labrum (Fig.1): pentagonal, symmetrical, trilobed, rugose, apical lobule serrated with small tubercles and with long slender setae; with 14-17 posterior setae; 6 long medial setae and 10 anteromarginal setae. Epipharynx (Fig. 3): pentagonal, with plegmatia, proplegmatium absent, epizygum absent, right zygum present, left zygum absent. Corypha with 4 blunt setae. Clithrum absent. Acroparia with long, slender, flat setae. Haptomerum with 2–3 sensillae and 4-6 heli in transverse row. Acanthoparia with 8 short sickle-shaped setae. Gymnoparia without setae. Chaetoparia with 2 defined rows of thick setae forming an arc. Pternotorma and dexiotorma well-developed, sclerotized, laeotorma less developed, without epitorma and apotorma. Laeotorma half length of dexiothorma. Haptolachus with 2 nesia. Crepis absent. Mandibles (Figs.4, 5): asymmetrical. Left cutting region with 3 teeth

and right with 2 teeth. Scrobe with 7-8 setae and 7-8 basolateral setae. Dorsal carina with 1 seta. Dorso-molar area with small group of short setae close to molar region. Left ventral process slightly smaller than right ventral process. Calyx not elongated. Brustia present. Acia well developed and wider than long. Stridulatory area absent. Ventral processes roughened. Maxilla (Fig. 6): with galea and lacinia separated; galea with uncus well developed; lacinia with 3 unci; maxillary palpus is very curved from a lateral view. Maxillary stridulatory area formed by row of 9-14 teeth (Fig. 8). Labium (Figs. 9-10): with hypopharyngeal sclerome asymmetric, strongly developed and sclerotized, left hypopharyngeal lobe with row of vertically oriented setae, proximal area of sclerome with 2 dense groups of setae. Glossa covered by slender setae and robust setae. Postmentum subtrapezoidal, with 4 slender, long setae. Posterior section of prementum with 4 setae, anterior section with 6 slender setae and 1 lateral seta close to labial palpus.

Thorax. Spiracles (T) (Fig.11): with C-shaped respiratory plate, concavity facing posteriorly, plate 0.12 mm long, 0.024 mm wide, with 6-8 irregular, large holes across diameter (Fig.13). Distance between 2 lobes of respiratory plate equal to diameter (dorso- ventrally) of bulla. Pronotum with lateral, scalloped scleromes, with an irregular shape, longer than wide, slightly separated and surrounded by 58–64 setae. Legs (Fig. 14): with size increasing gradually from prothoracic to metathoracic leg. Each tarsungulus with 2 proximal setae, tarsungulus of metathoraxcic leg smaller than preceding.

Abdomen. Abdominal spiracles (A) (Fig. 12): A1- 4 with C-shaped respiratory plate, A1- 3 of similar size (0.142-0.166 mm), A4 (0.071 mm) smaller than A1- 3, concavities of their respiratory plate facing anteriorly, A5- 8 (0.047mm) equal in size and conspicuously smaller, minimum concavities of their respiratory plate facing ventrally . Spiracular area with 6-12 short, medium-length and long setae. Dorsal segments of abdomen A2- 6 with 2- 5 rows of short, sharp setae and 1 row of slender, long setae. Raster (Fig.15): without palidia. Tegillum covered with long, flat setae. Anal opening Y-shaped. Dorsal and ventral

anal lobes densely covered with small, slender, acute, straight setae. Campus with 10-12 moderately long setae. Barbula with a few slender setae.

Diagnosis. The larva of A. mahdii has a Y-shaped anal opening and is without a palidium. The tegillum is covered by long, flat setae. Abdominal spiracles A1-A3 are of similar size and A5–A8 are equal in size but conspicuously smaller than the anterior spiracles and concavities of their respiratory plate facing ventrally. The apical antennomere have one dorsal sensory spot and two ventral sensory spots. The chaetoparia has two defined rows of thick setae forming an arc. The mandibles have a well developed acia, are longer than wide, and without a stridulatory area. Maxillary palpus is very curved from in lateral view. The galea and lacinia are separated. Each tarsungulus has two proximal setae.

The described larval characterists of A. mahdii correspond to the Melolonthinae and not to the known Glaphyridae, confirming the taxonomic placement of this species indicated by Hawkins 2006 and Smith et al., 2006.

ACKNOWLEDGMENTS

We thank Mario Elgueta, National Museum of Natural History (MNHN) Santiago - Chile, for the help in providing the identification of adult Scarabaeidae. We also thank Leticia Silvestre for her collaboration in the laboratory work.

REFERENCES CITED

Carne, P.B. 1951. Preservation techniques for scarabaeid and other insects larvae. Proceeding of the Linnean Society of New South Wales 76: 26-30.

Edmonds, W.D., and G. Halffter. 1978. Taxonomic review of immature dung beetles of the subfamily and Scarabaeinae (Coleoptera: Scarabaeidae). Systematic Entomology 3: 307-331. Evans, A.V., and A. B. T. Smith. 2009. An electronic checklist of the new world chafers (Coleoptera: Scarabaeidae: Melolonthinae) Version 3. Electronically published, Otawa, Canada. 353 pp. Available from: www-museum.unl.edu/research/entomology /SSSA /nwmelos.htm. Grebennikov, V.V., and C. H. Scholtz. 2004. The basal phylogeny of Scarabaeoidea ((Insecta : Coleoptera) inferred from larval morphology. Invertebrate Systematics 18: 321- 348. Hayes, W.P. 1929. Morphology, taxonomy and biology of larval Scarabaeoidea Illinois Biological Monographs 12(2): 1-125. Hawkins, J. S. 2006. A revision of the Chilean tribe Lichniini Burmeister, 1844 (Coleoptera: Scarabaeidae: Melolonthinae). Zootaxa 1266: 1-63. McQuillan, P.B. 1985. The identification of root feeding cockchafer lavae (Coleoptera: Scarabaeidae) found in pastures in Tasmania. Australian Journal of Zoology 33: 509-546. Peterson, A. 1960. Larvae of insects. An introduction to Neartic species. Part II. Coleoptera, Diptera, Neuroptera, Siphonaptera, Mecoptera, Trichoptera. Privately published, Culumbus, OH. 218 p. Ritcher, P. 1966. White grubs and their allies. A study of North American scarabaeoidea larvae. University of Oregon Press, Corvallis, OR. 219 p. Smith, A.B.T.; Hawks, D.C., and Heraty, J.M. 2006. An overview of the classification and evolution of the major scarab beetle clades (Coleoptera: Scarabaeoidea) based on preliminary molecular analyses. Coleopterists Society Monograph 5: 35- 46.

Figs. 1-8. Arctodium mahdii, third instar. 1) Head, frontal view; 2) Distal antennomere, a) lateral and b) ventral view; 3) Epipharynx; 4) Mandibles, ventral view a) left and b) right; 5) Mandibles, dorsal view a) right and b) left; 6) Maxillae, a) dorsal, b) ventral and c) lateral view; 7) Apex of lacinia and galea showing unci; 8) Maxillary stridulatory area. Bar = mm.

Figs. 9-15. Arctodium mahdii, third instar. 9) Labium, dorsal view; 10) Hypopharynx; 11) Thoracic spiracle; 12) Abdominal spiracle A1; 13) Respiratory plate; 14) Tarsungulus of legs a) prothoracic leg, b) mesothoracic leg, c) metathoracic leg, 15) Last abdominal segment, ventral view of raster. Bar = mm

5. DESCRIPCIÓN TAXONÓMICA DEL ESTADO LARVAL DE DOS ESPECIES DE SCARABAEIDAE: RUTELINAE: ANOPLOGNATHINI: BRACHISTERNINA.

Este artículo puede ser consultado bajo el título:

Cisternas, A. E. y Carrillo, Ll. R. DESCRIPTION OF LARVAE OF BRACHYSTERNINA Hylamorpha elegans (Burmaister, 1844) AND Aulacopalpus punctatus (Fairmaire and Germain, 1860) ASSOCIATED WITH CHILEAN PASTURES (COLEOPTERA: SCARABAEIDAE: RUTELINAE: ANOPLOGNATHINI). Accepted in The Colleopterist Bulletin (N° 1283)

DESCRIPTION OF THE LARVAE OF Hylamorpha elegans (Burmeister, 1844) AND Aulacopalpus punctatus (Fairmaire and Germain, 1860) (COLEOPTERA: SCARABAEIDAE: RUTELINAE: ANOPLOGNATHINI)

ABSTRACT

The third instar larvae of Hylamorpha elegans (Burmeister, 1844) and Aulacopalpus punctatus (Fairmaire and Germain, 1860) (Scarabaeidae: Rutelinae: Anoplognathini) are described. Descriptions are based on larvae reared from eggs laid by adults in the laboratory and larvae collected in the field. These are the first descriptions of anoplognathine larvae from the southern South America. The larvae feed in the root layer in pastures. The two species are easily distinguished by the coloration and roughness of the cephalic capsule and the distribution and shape of the setae of the raster.

RESUMEN

Se describe e ilustra el tercer estadio de Hylamorpha elegans (Burmeister, 1844) and Aulacopalpus punctatus (Fairmaire and Germain, 1860) (Scarabaeidae: Rutelinae: Anoplognathini). Las descripciones se basan en larvas criadas desde huevos puestos por

adultos en el laboratorio y desde larvas colectadas en el campo. Estas son las primeras descripciones de larvas de Anoplognathini del cono sur de América del Sur. Las larvas viven y se alimentan de la zona radical en las praderas del sur de Chile. Una de las formas de separar fácilmente ambas especies es a través de la coloración y rugosidad de la capsula cefálica y la distribución y forma de las setas del ráster.

INTRODUCTION

Hylamorpha elegans (Burmeister, 1844) and Aulacopalpus punctatus (Fairmaire and Germain, 1860) are members of genera that are endemic to southern South America. Difficulties in identifying species of larvae have delayed necessary advances in the ethology, biology and biological control. Likewise, lack of description and characterization of the preimaginal stages of larvae has impeded, for example, the establishment of effective systems to combat the damage as well as broader implications of insect-plant relationships. The scarab tribe Anoplognathini (Rutelini) includes over 150 species (Paucar-Cabrera and Smith 2002). Only four species of Anoplognathini in the larval stage have been described: two from Tasmania and Australia, Saulostomus villosus Waterhouse (McQuillan 1985) and Anoplognathus suturalis Boisduval (Hardy 1976), and two from the Neotropics, Platycoelia gaujoni Ohaus and Platycoelia lutescens Blanchard (Paucar-Cabrera and Smith 2002). Hylamorpha and Aulacopalpus are two genera endemic to southern South America that belong to the subtribe Brachysternina, a subtribe that is distributed in Chile and Argentina. Hylamorpha elegans is the most common species in various agroecosystems and with a wide distribution in Chile (Duran 1952; Carrillo and Cerda 1987; Ratcliffe and Ocampo 2002), although pastures are a common habitat (Aguilera et. al 1996). The genus is monotypic (Ratcliffe and Ocampo 2002), and adults are known as “San Juanes”, “pololos” or “pololo verde chico”. The distribution of Aulacopalpus punctatus is limited to southern regions of Chile (Valdivia south to Llanquihue) (Smith 2002), and its relative importance in the ecosystem is unknown. The genus includes nine species (Smith 2002), and adults are

known as “San Juanes”, “pololo café” or “pololo de otoño”. The larvae of these species develop in the soil where they feed on roots of plants, including crops. The objectives of this study were: 1) to describe the third larval state of H. elegans and A. punctatus and 2) to determine the specific morphological characters to distinguish the two species.

MATERIAL AND METHODS

The third instars used for the description of the two species were obtained by rearing larvae reared from eggs laid by femeles in the laboratory and by collecting larvae in the field for rearing to adulthood. The larvae were reared individually in plastic boxes with sieved soil. The material obtained was reared and maintained in an acclimatized chamber at 20 ± 2ºC, 60±5% RH without a photoperiod. The rearing boxes were examined weekly, with replacement of moisture lost over the intervening period and removal of any dead insects. Larvae of the third instar used for morphological descriptions were fixed in a solution of KAAD Mixture (ethyl alcohol, kerosene, glacial acetic and dioxane) (Carne 1951) and preserved in 70% alcohol. Morphological structures were mounted in Faure, Canada balsam and Hoyer solutions. Drawings were made using a stereomicroscope, a microscope, camera Lucida and microphotography. Measurements were taken with an ocular micrometer. Voucher specimens are deposited in the Entomology Collection of the Universidad Austral de Chile, Valdivia. The terminology of Ritcher (1966) was used in the description of the larvae. The parts of the larvae for description have also been used by Hayes (1929), Peterson (1960), Edmonds and Halffter (1978), Vanin and Costa (1980), McQuillan (1985), Paucar-Cabrera and Smith (2002), Mico and Galante (2003), Mico et al. (2003), Grebennikov and Scholtz (2004) and Vallejo and Moron (2008).

RESULTS

Hylamorpha elegans (Burmeister, 1844) Third instar (Figs. 1–14)

The description is based on larvae reared from eggs laid by adults and larvae collected in pastures and meadows in Valdivia and surrounding areas, such as the Santa Rosa Experimental Station of the Universidad Austral de Chile, in the Región de Los Rios, 39° 47` S and 73º 14` W, at 21 m elevation.

Description. C-shaped larvae. Head : Width of the head capsule L1: 1.57 ± 0.08 mm (n=20); L2: 2.62 ± 0.08mm (n=20) and L3: 4.20 ± 0.12 mm (n = 20). Cranium (Fig. 1): Ocellus absent. Surface slightly rugose, shining, reddish; frontal suture whitish and bisinuate, forming a sharp angle at the joint; epicranial suture 1/3 of the length of the frontal suture; 5-6 dorsoepicranial setae and 2 lateral long setae and 3 short setae on each side; 8-10 anterior frontal setae and 9 posterior frontal setae; 1 seta adjacent to the antennal socket; 2 setae over precoila; 8-9 long setae adjacent and lateral to the antennal socket when viewed from the front. Antenna (Fig. 2): Four antennomeres. Apical antennomere with 1 dorsal sensory spot and 2 ventral sensory spots. Second antennomere 1.3 times as long as antennomere 1. Third antennomere with an elongated process and a ventral sensory spot. Clypeus (Fig. 1): Trapezoidal. Surface of preclypeus smooth, lighter in color, without setae. Postclypeus slightly rugose with 2 exterior setae on each side and 2 clypeal setae. Labrum (Fig.1): Suboval, asymmetrical; anterior margin irregular, apical lobule with a pair of blunt setae, with 8-10 posterior setae, 6 medial setae, and 6 maginal aoical setae. Epipharynx (Fig. 3): Suboval, without plegmata, proplegmatium absent, epizygum sclerotized and not connected to the zygum. Corypha with 2 blunt setae and 2 slender and straight setae. Clithrum absent. Haptomerum with 3–4 sensilla a beak-like process and 8 coarse setae in transverse row. Acanthoparia with 10-12 short sickle-shaped setae. Gymnoparia without setae. Chaetoparia covered with straight setae. Pternotorma,

dexiotorma and laeotorma well developed and sclerotized, with epitorma and without apotorma. Dexiothorma 1.3 times as long as the laeotorma. Haptolachus with 2 nesia, sensory cone and plate; sensory cone smaller than pterotorma. Crepis absent. Mandibles (Figs. 4, 5): Asymmetrical. Left cutting region with 3 teeth and right with 2 teeth. Scrobe with 7-8 basolateral setae. Dorsal carina with 1 seta. Dorsomolar area with small group of short setae close to molar. Right ventral process smaller than left ventral process. Calyx not elongated. Brustia present. Acia well developed. Stridulatory area present, suboval with approximately 54-62 transverse ridges. Ventral processes with rough surface. Maxilla (Fig. 6): Galea and lacinia fused; galea with uncus well developed; lacinia with 2 unci fused at the base (Fig. 7). Maxillary stridulatory area formed by row of 9-10 acute, anteriorly directed, recurved teeth and 1 distal, blunt tubercle (Fig. 8). Labium (Figs. 9,10): Hypopharyngeal sclerome asymmetrical, strongly developed and sclerotized, left lobule of hypopharyngeal with row of horizontally oriented setae, proximal area of scleroma without setae. Glossa covered distally by slender, distal setae and posterior robust setae. Postmentum with 2 basal, slender setae and 2 apical, long setae. Basal section of prementum with 2 rows of setae; setae long and moderate in length; anterior section of prementum with 8-10 slender setae and 3 lateral setae close to labial palp.

Thorax. Spiracles (Fig.11): C-shaped respiratory plate, 0.35 mm long, 0.25 mm wide, with 20-23 irregular holes across diameter (Fig.12). Distance between lobes of respiratory plate 1.5X the diameter of bulla. Pronotum: With lateral, scalloped scleromata (yellow, irregularly shaped, surrounded by 10–12 setae). Dorsum of prothorax with 3 irregular rows of long, medium-length and short setae. Legs (Fig. 13): Length of legs increases gradually from prothorax to metathorax. Each tarsunguli with 2 proximal setae.

Abdomen. Abdominal spiracles A2-A8 and T of similar size (0.35-.0.37mm) A1 somewhat smaller in size than anterior ones (0.29 mm). Spiracular area with 7-12 short, medium-length, and long setae. Dorsal segments A2–A6 with 4-6 rows of short setae mixed with very long and long setae. Raster (Fig.15): Without palidia. Tegillum with 59-

71 hamate setae. C-shaped anal opening. Dorsal anal lobules covered distally with small, straight and cylindrical setae. Ventral anal lobes with slender, straight, medium-length and small setae. Campus with 8 moderately long setae. Barbula not well defined, with a few long and very long, slender setae.

Aulacopalpus punctatus (Fairmaire and Germain, 1860) Third instar (Figs.15-28)

The description is based on larvae reared from eggs laid by adults and larvae collected in pastures in the Región de Los Rios and Región de Los Lagos, from locations such as Rio Bueno, Osorno and Purranque. 40° 20` S and 72º 58` W, at 75 m elevation.

Description. C-shaped larvae. Head: Width of the head capsule L2: 3.34 ± 0.13 mm

(n=5) and L3: 4.8 ± 0.12 mm (n = 14). ). Cranium (Fig. 15): Ocellus absent. Surface rugose, shiny , reddish-brown; frontal suture white and bisinuate, forming a sharp angle at the joint; epicranial suture 1/4 of the length of the frontal suture; 10-12 dorsoepicranial setae and 10-12 lateral setae on each side; 18-20 anterior frontal setae and 24-26 posterior frontal setae; 5 setae adjacent to antennal socket; 16-18 setae over precoila; 8-9 long setae adjacent and lateral to antennal socket when viewed from front. Antenna (Fig.16): Four antennomeres. Apical antennomere with 1 dorsal sensory spot and 2 ventral sensory spots. Second antennomere 1.5 times as long as antennomere 1. Third antennomere with elongated process and ventral sensory spot. Clypeus (Fig. 15): Shape trapezoidal. Surface of preclypeus smooth, lighter in color, without setae. Postclypeus slightly rugose with 2 exterior setae on each side and 10 clypeal setae. Labrum (Fig.15): Shape suboval, asymmetrical; anterior margin irregular; apical lobe with 2 blunt setae; with 14-16 posterior setae; 6 medial setae and 6 anteriomarginal setae. Epipharynx (Fig.17): Suboval, without plegmata, proplegmatium absent, epizygum sclerotized and not connected to zygum. Corypha with 2 blunt setae and 2 slender straight setae. Clithrum absent. Haptomerum

without sensilla, with beak-like process and 8 coarse setae in transverse row. Acanthoparia with 5-7 short, sickle-shaped setae. Gymnoparia with short setae. Chaetoparia covered with straight setae. Pternotorma, dexiotorma and laeotorma well developed and sclerotized, with epitorma and without apotorma. Dexiotorma 1.3 times as long as laeotorma. Haptolachus with 2 nesia, sensory cone and plate; sensory cone equal in size to pternotorma. Crepis absent. Mandibles (Figs. 18, 19): Asymmetrical. Left cutting region with 4 teeth and right with 3 teeth. Scrobe with 10-12 basolateral setae. Dorsal carina with 2 setae. Dorsomolar area with small group of short setae close to molar. Right ventral process smaller than left ventral process. Calx not elongated. Brustia present. Acia well developed. Stridulatory area present, suboval with approximately 32-35 transverse ridges. Ventral process with rough surface. Maxilla (Fig. 20): With galea and lacinia fused; galea with uncus well developed; lacinia with 1 unci, and small tooth fused at base (Fig.21). Cardo with few setae. Maxillary stridulatory area formed by row of 10-12 acute, anteriorly directed, recurved teeth and 1 distal, blunt tubercle (Fig. 22). Labium (Figs. 23-24): Hypopharyngeal sclerome asymmetrical, strongly developed and sclerotized, left lobe with short row of horizontally oriented setae and proximal area of scleroma without setae. Glossa covered distally by slender setae and posterior robust setae. Postmentum with 4 posterior slender setae and 6 short and medium-length, anterior setae in the middle part and 4 long, anterior setae. Posterior section of prementum with 1 row of 16 very long and long setae and 1 row of 6 medium-length setae, anterior section of prementum with 2 very long and 8 slender and medium-length setae and 4 lateral setae close to labial palp.

Thorax. Spiracles (Fig. 25): C-shaped respiratory plate, 0.35 mm long, 0.18 mm wide, with 15-18 irregular holes across diameter (Fig.26). Distance between two lobes of respiratory plate = 1.5 times diameter of bulla. Pronotum: With lateral scalloped scleromas (yellow, irregularly shaped, surrounded by 14–16 setae). Dorsum of prothorax with 3 irregular rows of long, medium-length and short setae. Legs (Fig. 27): Length of legs increases gradually from prothorax to metathorax. Each tarsungulus with 2 proximal setae.

Abdomen. Abdominal spiracles A2-A7 of similar size (0.42-0.45 mm), A1 (0.36 mm)somewhat smaller size than A2-7 and A8 larger than anterior ones (0.48 mm). Spiracle area with 9-22 short, medium-length and long and very long setae. Dorsa of A2–A6 with 4-5 rows of short and straight setae mixed with long and very long setae. Raster (Fig.28): Without palidia. Tegillum with 190-200 short, straight and flat setae, as well as two clusters of long, slender and straight setae at each ends of anal opening. C-shaped anal opening. Dorsal anal lobes distally covered with small, straight and cylindrical setae. Ventral anal lobes with small, slender, straight and cylindrical setae. Campus with 8-10 moderately long setae. Barbula not well defined with few long and very long slender setae.

DISCUSSION

The larva of H. elegans has a C-shaped anal opening without palidia. The tegillum has 59- 71 hamate setae. Spiracles A2-A8 and T are of similar size, spiracles on A1 somewhat smaller size than the anterior ones. The apical antennomere has one 1 dorsal sensory spot and two ventral sensory spots. The respiratory plate of the thoracic spiracle has 20-23 irregular holes across its diameter. The maxillary stridulatory area is formed by a row of 9- 10 acute, anteriorly directed, recurved teeth and a distal, blunt tubercle. The cranium is slightly rugose and red in color. The mandibular stridulatory area is suboval with approximately 54-62 transverse ridges. The haptomerum with 3–4 sensilla, a beak-like process and eight coarse setae in transversal row. Please provide a comparison with A. punctatus. These species could be found in the same area, so a comparison would be helpful. The text above is a summary of the description. It is not a diagnosis that allows for easy separaton from other species.

The larva of A. punctatus has a C-shaped anal opening without palidia. The tegillum has 190-200 short, straight, flat setae, as well as 2 groupings of long, straight and slender setae at each end of the anal opening. Spiracles A2-A7 of similar size, A1 somewhat smaller size than those on A2-A7 and A8 larger than the preceding. The apical antennomere has 1

dorsal sensory spot and two ventral sensory spots. The respiratory plate of the thoracic spiracle has 15-18 irregular holes across its diameter. The maxillary stridulatory area is formed by a row of 10-12 acute, anteriorly directed, recurved teeth and a distal, blunt tubercle. The cranium rugose and reddish-brown. The mandibular stridulatory area is suboval with approximately 32-35 transverse ridges. The haptomerum is devoid of sensilla but has a beak-like process and eight coarse setae in a transverse row.

Characters of Anoplognathini larvae

Our descriptions of Hylamorpha and Aulacopalpus larvae brings the knowledge of world Anoplognathini larvae to six genera and species world-wide. The characters that we describe allow for a better and more certain character definition of the tribe. We add to the characters provided by Ritcher (1966), Hardy (1976), McQuillan (1985), and (Paucar- Cabrera and Smith 2002) for the tribe Anoplognathini and characterize the larvae as follows: Apical antennomere with one dorsal sensorial spot; slightly asymmetric labrum; haptomerum with or without a beak-like process and with a thin basal row of heli; mandible with a suboval ventral, stridulatory area and with transverse ridges; galea and lacinia fused forming the mala; maxillary stridulatory area with recurved teeth and a tubercle anterior to stridulatory teeth; straight or slightly curved right anal opening.

ACKNOWLEDGMENTS

We thank Mario Elgueta D., National Museum of Natural History (MNHN) Santiago-Chile, for the help in providing the identification of adult. We also thank Marcelo Villagra and Gustavo Valdebenito who collaborated with us in the field work and Leticia Silvestre for her collaboration in the laboratory work.

REFERENCES CITED

Aguilera, A., E. Cisternas, M. Gerding, and H. Norambuena. 1996. Plagas de las Praderas [pp. 309-339]. In: Praderas para Chile (I. Ruiz, editor). Instituto de Investigaciones Agropecuarias, Santiago, Chile. Carne, P. B. 1951. Preservation techniques for scarabaeid and other insects larvae. Proceeding of the Linnean Society of New South Wales 76: 26-30. Carrillo, R. and L. Cerda. 1987. Zoofitófagos en Nothofagus chilenos. Bosque 8:99-103. Duran, L. 1952. Aspectos ecológicos de la biología del San Juan Verde, Hylamorpha elegans (Burm.) y medición de las demás especies de escarabaeidos perjudiciales en Cautín. Agricultura Técnica 12: 24-36. Edmonds, W. D. and G. Halffter. 1978. Taxonomic review of immature dung beetles of the subfamily Scarabaeidae (Coleptera: Scarabaeidae). Systematic Entomology 3: 307- 331. Grebennikov, V. V., and C. H. Scholtz. 2004. The basal phylogeny of Scarabaeoidea (Insecta : Coleoptera) inferred from larval morphology. Invertebrate Systematics 18: 321- 348. Hardy, R. J. 1976. Observations on the pasture beetle, Saulostomus villosus Waterhouse (Scarabaeidae: Rutelinae). Journal of the Australian Entomological Society 15: 281-284. Hayes, W. P. 1929. Morphology, taxonomy and biology of larval Scarabaeoidea Illinois, Biological Monographs 12(2): 1-125. McQuillan, P. B. 1985. The identification of root feeding cockchafer lavae (Coleptera: Scarabaeidae) found in pastures in Tasmania. Australian Journal of Zoology 33: 509-546. Mico, E., and E. Galante. 2003. Biology and new larval description for three cetoniine beetles (Coleoptera: Scarabaeidae: Cetoniinae: Cetoniini: Cetoniina: Leucocelina). Annals of the Entomological Society of America 96 (2): 95-106. Mico, E., Moron, A. and E. Galante. 2003. New larval description and biology of some new world Anomalini beetles (Scarabaeidae: Rutelinae). Annals of the Entomological Society of America 96 (5): 597-614.

Paucar-Cabrera, A. and A.B.T. Smith. 2002. Larval descriptions for the Neotropical genus Platycoelia (Coleoptera: Scarabaeidae: Rutelinae: Anoplognathini). The Coleopterists Bulletin 56 (3): 438-445. Ratcliffe, B., and F. Ocampo. 2002. A review of the genus Hylamorpha Arrow (Coleoptera: Scarabaeidae: Rutelinae: Anoplagnathini: Brachysternina). The Coleopterist Bulletin 56:367-378. Ritcher, P. 1966. White Grubs and Their Allies. A study of North American Scarabaeoidea larvae. University of Oregon Press. Corvallis, OR. Smith, A.B.T. 2002. Revision of the southern South American endemic genus Aulacopalpus Guerin-Méneville with phylogenetic and biogeographic analyses of the subtribe Brachysternina (Coleoptera: Scarabaeidae: Rutelidae: Anoplognathini). The Coleopterist Bulletin 56: 379-437. Vanin, S. A., and C. Costa. 1980. Larvae of Neotropical Coleoptera III: Scarabaeidae, Rutelinae. Papéis Avulsos de Zoología. 33(17): 275-283. Vallejo, F., and M. Morón. 2008. Description of the immature stages and redescription of the adults of Ancognatha scarabaeoides Erichson (Coleoptera: Scarabaeidae: Dynastinae) a member of the soil assemblage in Colombia. The Coleopterists Bulletin 62: 154-164.

0.5

3 1.0 2 1 1.0

4a 4b 6a 6b 1.0 1.0

0.5 7 5a 5b 8

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Figs. 1-8. Hylamorpha elegans, third instar. 1) Head, frontal view, distal antennomere dorsal view ; 2) Distal antennomere lateral view; 3) Epipharynx; 4) Mandibles, ventral view, a) left and b) right; 5) Mandibles, dorsal view, a) right and b) left); 6) Maxillae, a) dorsal view and b) ventral view; 7) Apex of mala showing unci; 8) Maxillary stridulatory area. Scale bar in mm.

9 0,5 10 0,5 11 0,2

13a 0,1 12 13b 13c 0,2

1.0 Figs. 9-15. Hylamorpha elegans, third instar. 9) Labium dorsal view; 10) Hypopharynx; 11) Thoracic spiracle; 12) Respiratory plate; 13) Tarsungulus of a) prothoracic leg, b) mesothoracic leg and c) metathoracic leg 14) Last abdominal segment, ventral view of raster. Scale bar in mm.

Figs.15- 22. Aulocopalpus punctatus, third instar. 15) Head, frontal view; 16) Distal antennomere, a) ventral , b) dorsal and c) lateral view; 17) Epipharynx; 18) Mandibles, ventral view a) left t and b) righ; 19) Mandibles, dorsal view, a) right and b) left ;20) Maxillae, a) dorsal and b) ventral view; 21) Apex of mala showing unci; 22) Maxillary stridulatory area. Scale bar in mm.

Figs. 23-29. Aulocopalpus punctatus, third instar. 23) Labium dorsal view; 24) Hypopharynx; 25) Thoracic spiracle; 26) Respiratory plate; 27) Holes of respiratory plate; 28) Tarsungulus of; a) Prothoracic leg; b) Mesothoracic leg; c) Metathoracic leg; 29) Last abdominal segment, ventral view of raster. Scale bar in mm.

6. SELECCIÓN DEL SITIO DE OVIPOSICIÓN DE Hylamorpha elegans (Burm.) (SCARABAEIDAE: RUTELINAE).

Este artículo puede ser consultado bajo el título:

Cisternas, A. E. y Carrillo, Ll. R. 2010. OVIPOSITION SITE SELECTION BY THE SCARABAEID BEETLE Hylamorpha elegans (Burm.) (COLEOPTERA: SCARABAEIDAE: RUTELINAE) IN RESPONSE TO PLANTS, SOIL TYPE AND COVERS. Australian Journal of Entomology. ID: AEN-OA-Jan 2011-4165 (submitted)

OVIPOSITION SITE SELECTION BY THE SCARABAEID BEETLE Hylamorpha elegans (Burm.) (COLEOPTERA: SCARABAEIDAE: RUTELINAE) IN RESPONSE TO PLANTS, SOIL TYPE AND COVERS.

Ernesto Cisternas1* and Roberto Carrillo2 1 Graduate School, Faculty of Agricultural Sciences, Universidad Austral de Chile 2 Laboratory of Entomology, Institute of Production and Plant Protection, Universidad Austral de Chile, P.O. BOX 567 Valdivia, Chile. * Corresponding author email: [email protected]

Abstract

We studied the effect of soil, soil covers and plants on the oviposition preference of Hylamorpha elegans (Burm.), a Chilean polyphagous scarabaeid. H. elegans females favor soils with a greater amount of clay and loam particles than sand and sawdust for ovipositing. Maternal H. elegans preferentially oviposited in soils with plants. Beetles were able to respond to aerial plant cues, increasing their oviposition in soils with plants or dummy plants, where they were able to assess the stimulus quantity. Also, they were able to assess root stimuli, increasing their oviposition site selection where plant roots were present; in this case, the response was also related to the quantity of the stimulus. Visual cues such as oviposition stimulus play an essential role in the pre-alighting period, while physical and chemical cues increase in importance in the post-alighting period. Results confirm that even polyphagous insects respond to general abiotic and biotic environmental cues to maximize offspring performance, a product of selection pressure.

Key words Scarabaeidae, Rutelinae, Hylamorpha elegans, oviposition site selection.

INTRODUCTION

The white grub H. elegans is a polyphagous insect pest which feeds on the roots of berries, cereal crops, pastures and other crops in Chile (Duran 1976; Klein & Waterhouse 2000). Both the biology and ecology of this species have been widely reviewed by Rivera, (1904); Durán, (1952) and Carrillo, (1986).

Selecting a suitable oviposition site, is vital for the survival of the progeny in most phytophagous insects because a good site, minimizes threats and maximizes egg survival and growth potential. Studies have shown that both abiotic and biotic factors can alter oviposition site selection in different taxa, including insects. Such factors include the presence of predators and predator cues (Blaustein et al. 2004; Brodin et al. 2006), the presence of conspecifics (Ulmer et al. 2003; Sugiura et al. 2007), soil moisture conditions (Pacchioli & Hower 2004; Ward & Rogers 2007), plant phenology (Pierce & Gray 2006) soil conditions (MacDonald & Ellis 1990; Simelane 2007) and chemical cues (Grant et al. 2000).

Oviposition site selection involves benefits, such as the improvement of offspring survival and growth (Kiflawi et al. 2003), but also costs, like the time needed to find hosts, decreased hatchability etc. (Hirayama & Kasuya 2010). Therefore, there is a trade-off between benefits and costs in the selection of an oviposition site.

Some species of adults Scarabaeid are able to select the oviposition site. Popillia japonica Newman oviposition behavior is selective and it is influenced by plant derived cues, which are evaluated before and after digging into the soil. This species can also discriminate between the quantity and quality of artificial cues (Szendrei & Isaacs 2005). Régnière et al. (1981) and Allsopp et al. (1992), who worked with the same species oviposition responses, found that females selected certain soils based on qualities such as texture, moisture and organic matter content. This selective oviposition site behavior could

be important for scarabaeids because, in spite of their polyphagous habits, habitat quality strongly influences their overall reproductive success because factors affecting larval performance are highly variable both temporally and spatially (Rosentarits 1996). Reproductive strategies involving minimal post oviposition parental care and limited larvae dispersal, as is the case of the scarabaeid, should be utilized by the females that choose oviposition sites in order to maximize larval performance. However, in some phytophagous scarabaeids, such as Costelytra zealandica (White) and Heteronychus arator (Fabr.), adults did not respond to plant species, plant density or plant height (East & King 1977).

Our objective was to examine how soil type, soil substrates and plant material influence H. elegans oviposition site selection. We assessed the attractiveness of plant and soil characteristics to adult H. elegans. In order to evaluate visual cues we investigated the response of H. elegans oviposition site selection to plants and dummy plants and their response to the quantity of the stimuli.

MATERIALS AND METHODS

Insects. Adult H. elegans were collected in the field at the Universidad Austral de Chile’s Santa Rosa Experimental Station (39°47’S and 73°14’ W) and near Pilmaiquen (40°39’S and 72°39’W). Each insect was sexed according to their external characteristics (Cisternas 1986). The adult insects were conserved for 24 hrs. in plastic containers (10 L) with foliage of Nothofagus obliqua (Mirb.) Oerst. before being released into cages. For the field experiment 400 adults at a sexual rate of 1:1 were released in each cage. On the other hand, in the laboratory experiments a single fertilized female was placed into the experimental arena of each cage. During the experimental period the N. obliqua foliage in the cages was changed three times a week.

Field oviposition arena. Two cages of white netting (antiafide 16/10 40 mesh, TenaxR) measuring 22 m3 (6 x 2 x 1.85 m) were installed at the Universidad Austral de Chile’s Santa Rosa Experimental Station (39°47’22’’S and 73°14’01’’W). The cage floor was covered with black plastic 2 mm thick (Filmoamérica ,Brasil). Treatments were established making 80 equidistant holes, with steel cores (10x10cm). Each hole was lined with an empty plastic pot (Total Pack® 14 Argentine) of 1.4 L. Inside this a pot of similar texture, size and volume was placed. The pots with treatments were weighed to uniform weights. Three times a week three pots from each treatment were removed and weighed to establish their moisture conditions; water was added if necessary to maintain a uniform moisture for all the treatments.

Soil and substratum. The soil used was collected in different locations in the Lake and River Regions. 1. Inceptisol (Huiño Huiño, soil series), Aquandic Humaquept: (40°43’S and 72°48’W) 139 m elevation. 2. Ultisol (Huilma, soil series), Andic Palehumults: (40°47’S and 73°16’W) 108 m elevation. 3. Andisol (Puerto Fonk, soil series), Pachic Melanudands: (40°39’S and 72°39’W) 219 m elevation y 4. Andisol (Valdivia, soil series), Duric Hapludands: (39°45’ S 73°14’ W) 13 m elevation (Ciren, 2003). The substratum were: 1. River sand and 2. Sawdust (> 90 % N. obliqua).

Plants and roots. For the plant treatments in both seasons we used 3 g of ryegrass seed Lolium multiflorum Lam. cv. Tama for each pot. For the root treatment in the first season Lolium spp (>90 %) roots were field collected. Each treatment had 1 cm long roots which were washed and covered with a layer of soil 1 cm deep. In the second experimental year, roots were obtained in a different way because the experiment was set up and 3 g of ryegrass cv. Tama were sown 20 days before the bioassays started. The day before the start of the experimental phase, the plants’ foliage was cut below the soil level and covered with 1 cm of soil.

Covers. The soil cover studies consisted of: 1. chopped oat straw, 2. grated cow manure 3. sawdust (> 90 % N. obliqua) and 4. bare ground. The soil used in these experiments was from the Valdivian Andisol Series. A layer of 2 cm deep cover was applied on the soil (Figure 1).

Laboratory oviposition arena. Bioassays were carried out in a cage made with a plastic box (Wenco®) 35 x 24 x 13 cm installed over a base of 100 mm of Aislapol ® with adensity of 10 kg mˉ² (Basf Group, Chile) (external dimensions of 50 x 30 x 10 cm of height). Four oviposition arenas with diameters of 7.5 cm were made at the center of each cage. White plastic glasses of 300 mL (Mater, Chile) were used as pots. The soil used was an Andisol from the Valdivia Series with a humidity of 35 ± 5 % w/w. The treated soil blocks were installed in a cage in random positions. All arenas were placed in the environmental chamber at 20 ± 2 ºC, 60 ± 5 % RH and 16:8 (L:D) for 35 to 40 days.

Bioassays

Experiment 1. To determine H. elegans oviposition response to the density of artificial stems 6.5 cm long and 2 mm wide wooden toothpicks (Marco Polo® ICB S.A.) were used. The densities were: bare ground (0); 1133.5 (5); 2267 (10) and 4534 (20) wooden sticks m ˉ2 (wooden stick pots ˉ¹). Each wooden stick was buried 1 cm in the soil. Twenty arenas were established for this experiment.

Experiment 2. To determine H. elegans oviposition response to the density of L. multiflorum cv. Tama roots, the treatments were sown 20 days before the beginning of the experiments. The foliage plants were cut below the soil level and covered with 1 cm of soil. The treatments were: bare ground (0) g roots; 0.023±0.04 g roots DW potˉ¹ equivalent of 0.1g of seeds (23 seeds potˉ¹); 0.104±0.02 g roots DW pot-1 equivalent of 0.5 g of seeds (115 seeds potˉ¹) and 0.188±0.03 g roots DW /potˉ¹ equivalent of 1g of seeds (230 seeds potˉ¹). Twenty arenas were established for this experiment.

Experiment 3. H. elegans response to roots and dummy plants was assessed using roots 0.188±0.03 g DW pot ˉ¹ and dummy plants 4534 toothpicks mˉ2 Plants were sown and the foliage area was removed and covered with soil 1 cm deep before the start of the experiment. The treatments were: without roots/ without artificial stems; with roots/with artificial stems; without roots/with artificial stems and with roots/without artificial stems. Twenty arenas were established for this experiment.

Assessment of oviposition

The number of eggs and larvae found in each soil block was recorded. In the field experiment and laboratory bioassays each soil block was strained (16, 9 and 4 mesh) with a sieve, U.S. Standard Sieve Series, Soiltest,Inc. and manually inspected. L1 larvae were collected with forceps and eggs with a paintbrush between 35 and 40 days post released adult.

Statistical analysis

The field experiments had a randomized block design in a factorial arrangement (5x2), 5 soils or substratum with and without plants. The experiment with soil covers used the same experimental design, 4 covers with and without roots. Each treatment had 8 replicates and was evaluated using an ANOVA test (PROC GLM, SAS Institute 2009). The laboratory experiments evaluated the average number of insects per soil block and then that number was compared to other treatments estimating the oviposition ODD ratio using logistic regression (PROC Logistic, SAS Institute 2009). The critical value used to establish the significance of all these tests was determined at P ≤ 0.05.

RESULTS

Field experiment

Effect of soil type, sawdust, sand and plant presence on oviposition selection sites Fig. 2 displays how soil type affected female H. elegans selection of oviposition sites averaging two seasons. Females laid significantly fewer eggs in the sand and sawdust when compared to the other soil types However, no differences were found between sand and sawdust. Nonetheless, the differences among soil types were significant (F= 18.50, P<0.0001). When plants were present female H. elegans were more likely to choose the site for oviposition in andisol, ultisol and inceptisol (F=60.22, P<0.0001). There were interactions between soil and plants, (F=7.98, P<0.0001) but not between sand or sawdust with plants. In both experimental seasons the number of eggs and offspring did not increase with the presence of plants in sand.

Effect of soil covers and plant roots on the oviposition site selection of H. elegans The results showed that in both seasons the soil covers affected the oviposition behavior of H. elegans (Fig. 3, 4). The effect was significant (F=4.36, P<0.0033 and F=4.57, P<0.0062), for first an second season respectively. Overall, the females showed a preference for pasture in the first season but in the second season manure choice was statistically different for all covers. This may have occurred due to the differences in composition and age of the manure used. In both years the presence of roots increased the oviposition, however, the increased response was higher during the second year, 3.71 times vs 2.31 times, but in both seasons, plant roots and his interaction with covers were not significant. This was probably due to the way in which roots were added to the soil; in the first year roots were placed as a layer covered with soil, and in the second year roots emerged from living plants seeded in pots, whose foliage was removed and roots covered with 1cm of soil, (Fig.1).

Laboratory experiment

The use of visual cues by female H. elegans selecting oviposition sites Females use visual cues to select oviposition sites. When wooden sticks 6.5 cm high were used as dummy plants, in general, females increased the number of eggs oviposited (Fig. 5), with an increase in the density. No eggs were deposited in bare soils. The relationships between dummy plant densities and egg oviposition were significant (P <0.05).

Effect of chemical cues on oviposition behavior To measure the effect of chemical cues on the oviposition behavior of female H. elegans, different root biomass were applied to each pot. There were significant differences in the response of females to root biomass (Fig. 6). The response increased with the amount of biomass. In fact, this was the most significant response, being all the treatments with plant biomass (roots) statistically different from the control (P <0.05).

Importance of visual and chemical cues on the selection of oviposition sites by female H. elegans H. elegans response to visual cues when selecting an oviposition site was more significant than its response to chemical cues (Fig.7). Significantly more eggs were found in soils with dummy plants than in soils with roots (P <0.05). However, there was a positive interaction between visual (dummy plants) and chemical cues (roots).

DISCUSSION

There were clear differences in how female H. elegans responded to physical and chemical environmental cues. These differences are, in general terms, similar to those observed in other underground phytophagous insects of the same family (Szendrei & Isaacs 2005) and other insect groups (Pacchioli & Hower 2004).

Physical characteristics of the soil influenced the oviposition choice of female H. elegans because they avoided soils with sand and sawdust. The findings of this research are consistent with previous studies that have shown that sandy soils are unsuitable for root feeding insects. There are most likely two main reasons for females to reject this type of soil. First of all, the abrasive effect of the sharp edges of sand particles can directly affect females behavior (Hoback & Golick 2000) and secondly, due to a genetic selection (Thompson & Pellmyr 1991) that has favored the selection sites that facilitate the growth and survival of the neonate larvae offspring (Thompson 1988; Holland et al, 2004). Sandy and other rapidly drying soils are unsuitable for the larval survival of root feeding insects ( MacDonald & Ellis 1990; Brust & House 1990). The survival of immature offspring was reduced in Diptera when sawdust and sand were used as substrates, because they also dry rapidly (Schmidtmann 1991).

The differences observed in the response to sawdust between the two experimental years could be related to the physical or chemical characteristics of the sawdust used, because factors such as time from its elaboration, type of material used for its preparation, and storage conditions can affect its properties. In this case sawdust came from the same species, N. obliqua, but in the second experimental year it had one year of storage. A decrease in females response occurred in the second season in both experiments in which sawdust was the only substrate and in those where it was used as a cover (Fig. 3). This lack of response in the second season may have been due to the loss of chemical substances during the storage which may have affected female behavior.

The absence of differences between soil types can be explained because all the soils studied are rich in clay and loam (Table 1), which has normally been the preferred soil particles by underground feeding insects because these have high water retention and little abrasive effect. In some cases underground phytophagous insects prefer clay rich soils because these soils readily form cracks and have better structure which is especially important for soil tunneling insects (Simenale 2007). In this case, because the soil was

pulverized when packed into the pots and then compacted to obtain a uniform compactness, these factors, which affect the female oviposition and larvae survival, were made uniform (Mac Donald & Ellis 1990; Simenale, 2007). In addition, the differing moisture retentions of the soils were reduced because soils were watered weekly. It is likely that under field conditions, using undisturbed soils with a continuity of pores, one could expect a different response from the females.

Female H. elegans were able to discriminate between bare soils, sand and sawdust with and without plants (ryegrass), increasing their oviposition in pots with plants. This indicates that plants provide oviposition cues to females, as occurs in other scarabaeid P. japonica (Szendrei & Isaacs 2005). Because plants can provide females with physical, chemical and visual cues experiments were carried out to study the effects of both chemical and visual cues. Artificial grass stems (sticky woods), received more eggs than bare ground demonstrating the primary role that visual stimulus plays as an oviposition cue. Moreover, female H. elegans were able to discriminate in relation to the stimulus quantity, generally increasing the oviposition in pots with high artificial stem density. To determine the effect of chemical cues, ryegrass roots were used, and there was a high oviposition site selection by female H. elegans in sites with increased number of roots in comparison to bare soil. Also, in this case, females were able to determine the quantity of stimulus available. When both visual and chemical stimuli were tested in combination or alone, the visual stimulus was preffered. The results suggest that female H. elegans select the oviposition site by using visual stimuli as pre-alighting, pre-ovipositional cues, while the chemical stimuli can act as post-alighting, pre-ovipositional cues. Our results do not allow us to indicate if the stimulus provided by the plant roots can be considered as root mediated information (semiochemicals, microbes, etc) or simply as a feeding resource that stimulates the female oviposition once the species is buried.

The effect of soil covers on oviposition site selection by H. elegans showed great differences between years within the same treatment. However, in general terms, there was

an increase in the oviposition when manure and oat straw was used as cover, probably because it increased the amount of moisture in the oviposition selection site due to the absorption of water by the manure and oat straw. The moisture content of the substrate has been shown to be fundamental in the selection of sites by scarabeids (Ward & Rogers 2007).

Our results corroborate that polyphagous species such as H. elegans carefully select their oviposition site. Their selection is not influenced by any specific stimuli (vertical objects, presence of plant roots, and soil texture) which is common among many different polyphagous species. Johnson et al. (2006) has indicated that oviposition response in one curculionidae was influenced by shoot, root and soil mediated information, the same as in this study. The reason for this selection was the very limited potential for relocation of the first instar larvae, due to its small size and limited capacity to burrow long distances; this could explain the oviposition site selection of female H. elegans. The preference- performance hypothesis proposed by Jaenicke (1978) predicts a strong selection pressure on adults to select oviposition sites where offspring fitness would be maximized (Szendrei & Isaacs 2005), a situation that has occurred in this species according to the results obtained.

ACKNOWLEDGEMENTS

We are grateful to Sonia Santana H., Jorge Barría M. and Manuel Muñoz owners of the farms where soil samples were obtained. We wish to thank to the Soil Laboratory personal at the Universidad Austral de Chile. We also thank Gustavo Valdebenito for his assistance in the field. We would like to thank Christine Harrower for her constructive criticism of the manuscript’s English writing and Hector Uribe for statistical advice.

REFERENCES

Allsopp PG, Klein. MG & Mc Coy EL. 1992. Effect of soil moisture and texture on oviposition by the japanese beetle and rose chafer (Coleóptera:Scarabaeidae). Journal of Economic Entomology 85, 2194-2200. Blaustein L, Klifawi M, Eitam M, Mangel M & Cohen JE. 2004. Oviposition habitat selection in response to risk of predation: Mode of detection consistency across experimental venue. Oecologia 138, 300-305. Brodin T, Johansson F & Bergstein J. 2006. Predator related oviposition site selection of aquatic beetles (Hydrophorus spp) an effects on offspring life history. Freshwater Biology 51, 1277-1285. Brust GE & House GJ. 1990. Effects of soil moisture, no tillage and predators on Southern corn rootworm (Diabostrica undecimpunctaTA Howard): survival in corn agroecosystem. Agriculture Ecosystem Environment 31, 199-216. Carrillo R. 1986. Plagas en praderas. In: Latrille, L. (ed.). Producción de forrajes. Universidad Austral de Chile, Facultad de Ciencias Agrarias. Instituto de Producción Animal. Valdivia, Chile Serie B-ll pp. 76-94. Ciren 2003. Descripciones de suelos, materiales y símbolos. Estudio agrológico X Región. Santiago de Chile, Chile. Publicación CIREN N° 123 Tomo I y II . 412 p. Cisternas AE. 1986. Descripción de los estados preimaginales de escarabaeidos en praderas antropogénicas de la zona sur de Chile. Tesis Lic. Agr., Valdivia. Universidad Austral de Chile, Facultad de Ciencias Agrarias 119 p. Durán L. 1952. Aspectos ecológicos de la biología del San Juan Verde Hylamorpha elegans (Burm.) y mención de las demás especies de especies de escarabeidos perjudiciales en Cautín. Agricultura Técnica 12, 24-36. Durán L. 1976. Problemas de la entomología agrícola en Chile Austral. Agro Sur 4, 119- 127. East R & King PD. 1977. Effect of botanical composition of pastures on insect pest populations. The New Zealand Entomologist 6, 273-278.

Grant GG, Zhoo B & Langevin D. 2000. Oviposition response of spruce budworm (Lepidoptera:Tortricidae) to aliphatic carboxylic acids Environmental Entomology 33, 119-127. Hirayama H & Kasuya E. 2010. Cost of oviposition site selection in a water strides Aquarius paludum insularis, egg mortality increases with oviposition depth. J.Insect Physiol. 56, 646-649. Hoback WW & Golick DA. 2000. Salinity and shade preferences between sympatric tiger beetle species Ecological Entomology 15, 180-187. Holland JN, Buchanan AL & Loubeau R. 2004. Oviposition choice and larval survivasl of an obligately pollinating granivorous moth. Evolutioinary Ecology Research 6, 607- 618. Jaenicke J. 1978. On optimal oviposition behavior in phytophagous insects. Theoretical Population Biology 14, 350-356. Johnson SN, Nicholas A, Birch E, Gregory PJ & Murray PJ. 2006. The “mother knows best” principle: should soil insects be included in the preference-operformance debate?. Ecological Entomology 31, 395-401. Kiflawi M, Blaustein L & Mangel M. 2003. Oviposition habitat selection in a mosquito Culiseta longiareolata in response to risk of predation and conspecific density. Ecological Entomology 28, 168-173. Klein C & Waterhouse D. 2000. Distribution and importance of arthropods associated with agriculture and forestry in Chile. ACIAR Monograph N° 68 231 p. Mac Donald P & Ellis CR. 1990. Survival time of unfed first instar western corn rootworm (Coleoptera:Chrysomelidae) and the effects of soil type, moisture and compactation on their mobility in the soil. Environmental Entomology. 19, 666-671. Pacchioli M & Hover A. 2004. Soil and moisture effects the dynamics of early instar clover root curculio (Coleoptera:Curculionidae) and biomass of alfalfa root nodules. Environmental Entomology 33, 119-127.

Pierce C & Gray M. 2006. Western Corn Rootworm Diabrotica virgifera Le Conté (Coleóptera:Chrysomelidae), Oviposition: A Variant's Response to Maize Phenology. Environmental Entomology 35, 423-434. Régnière J, Raab RL & Stinner RE. 1981. Popillia japonica: Effect of soil moisture and texture on survival and development of eggs and first instar grubs. Environmental Entomology 10, 654-660. Rivera M. 1904. Biología de dos coleópteros chilenos cuyas larvas atacan al trigo. Revista Chilena de Historia Natural 8, 241-254. Rosentarits WJ Jr. 1996. Oviposition site choice and life history evolution. American Zoologist 36, 205-215. SAS Institute 2009. Version 9.1.3. SAS Institute, Inc, Cary, NC. Schmidtmann ET. 1991 Suppressing immature house and stable flies in outdoor hutches with sand, gravel and sawdust bedding. Journal of Dairy Science 74, 3956-3960. Simelane D. 2007. Influense of soil textura, Moisture and Surface Crack son the Perfomance of a Root-Feeding Flea Beetle Longitarsus bethae (Coleoptera: Chrysomelidae) a biological control agent for Lantana camara (Verbenaceae). Environmental Entomology 36, 512 – 517. Sugiura S, Yamazaki K & Yamaura Y. 2007. Intraspecific competition as a selective pressure on the choice of a oviposition site in a phytophagous insect. Biological Linnean Society 92, 641-650. Szendrei Z & Isaacs R. 2005. Do plant cues influence the oviposition behavior of Japanese beetles? Entomologia Experimentalis et Applicata 117, 165-174. Thompson JN. 1988. Evolutionary ecology of the relationship between oviposition preference and performance of offspring in phytophagous insects. Entomologia Experimentalis et Applicata 47, 3-14. Thompson JN & Pellmyr O. 1991. Evolution of oviposition behaviour and host preference in Lepidoptera. Annual Review of Entomology 36, 69-89.

Ulmer B, Gillot C & Erlandson M. 2003. Conspecific eggs and Bertha Armyworm, Mamestra configurata (Lepidoptera:Noctuidae), oviposition site selection. Environmental Entomology 32, 529-534. Ward A & Rogers J. 2007. Oviposition response of scarabaeids: does ‘mother know best’ about rainfall variability and soil moisture? Physiological Entomology 32, 357-366.

Table 1. Soil texture of the tested soils

Clay Silt Sand Soil Type Texture % Andisol 18.6 63.9 17.5 Silt loam Inceptisol 27.0 64.3 8.7 Silt loam Ultisol 34.9 54.3 10.8 Clay silt loam

Fig. 1. Roots location in the soil cover experiments.

Cover cover Soil soil Soil

roots

soil

First season Second season

Fig. 2. Oviposition preference of H. elegans for different soil types, sand, sawdust and plants, average for two season.

120 A 100 B B 80 a 60 a a 40 b C 20 C b b

Average number/pots of insects Average a a a a 0 Sand Ultisol Inceptisol Andisol Sawdust

Without plant With plant

Fig. 3. Effect of soil covers and roots on the oviposition site selection of H. elegans. First season

60 A 50

40 B 30 B 20 B B

10 Average number/ of insects pot Average

0 Bare ground Sawdust Manure Oat straw Pasture

Without roots With roots

Fig. 4. Effect of soil covers and roots on the oviposition site selection of H. elegans. Second season.

50 A 40

30 B 20 B B

10 Average number/ of insects pot Average

0 Bare ground Sawdust Manure Oat straw

Without roots With roots

Fig. 5. Effect of different densities of dummy plants on H. elegans oviposition site selection.

25 a

20 b 15 b 10

Average number of insects/pot Average c

0 0 5 10 20 Dummy plant density (number/pot)

Fig. 6. Effect of biomass dosage on the ovipositon selection site behavior of H. elegans.

30 a 25

20 a 15 a 10 b

5 Average number of insects/pot Average

0 0 0.1 0.5 1 Roots density (g of seeds / pot)

Fig. 7. Effect of visual and chemical cues on the selection of oviposition sites by female H. elegans. WR: Without roots; WF: Without foliage; R: Roots; F: Foliage

5 Average number of insects/pot Average

0 WR/WF WR/F R/WF R/F

7. SELECCIÓN DEL SITIO DE OVIPOSTURA DE Phytholaema herrmanni Germain (COLEOPTERA: SCARABAEIDAE: MELOLONTHINAE).

Este artículo puede ser consultado bajo el título:

Cisternas, A. E. y Carrillo, Ll. R. ENVIRONMENTAL FACTORS THAT DETERMINE THE SELECTION OF OVIPOSITION SITE OF THE POLYPHAGOUS SCARABAEID Phytholaema herrmanni Germain (COLEOPTERA: SCARABAEIDAE: MELOLONTHINAE). Entomologia Experimentalis et Applicata EEA-2011-0007 (Submitted)

ENVIRONMENTAL FACTORS THAT DETERMINE THE SELECTION OF OVIPOSITION SITE OF THE POLYPHAGOUS SCARABAEID Phytholaema herrmanni Germain (COLEOPTERA: SCARABAEIDAE: MELOLONTHINAE)

ABSTRACT

We studied the effect of soil type, ground cover and plants on the oviposition preference of Phytholaema herrmanni Germain, an endemic Chilean polyphagous scarabaeid. The females of P. herrmanni do not have a soil type preference, rather selecting sites on the basis of the presence of plants. The females responded to the high density of dummy plants, showing the ability to quantitatively measure the stimulus. The females were also able to measure the stimulus of roots quantitatively, with very high level of selection of oviposition sites where plant roots were present. The response was particularly related to the quantity of the stimuli. Chemical signals as stimulus for oviposition of P. herrmanni plays a fundamental role in the post-alighting selection of an oviposition site. It was evident that the interaction between chemical and physical cues determines the selection of oviposition sites.

Key words: Scarabaeidae, Melolonthinae, Phytholaema herrmanni, oviposition

INTRODUCTION

The white grub P. herrmanni is a polyphagous insect pest feeding on roots found in pastures, cereals, berries and other crops in Chile (Durán, 1954; Durán, 1976; Aguilera et al., 1996; Klein & Waterhouse, 2000) The biology and ecology of this species has been widely reviewed by Rivera (1904); Durán, (1954); Durán, (1976); Carrillo, (1986). Information on the oviposition behavior and the selection of oviposition sites among Chilean species of scarabaeids is scarce. Rivera (1904) and Durán, (1954) indicate that for P. herrmanni flight is crepuscular at a low height and occurs in evening hours. The adult does not ingest food and ovipositing females look for soil with vegetation for oviposition. Works related to the selection of sites and vegetation types by East & King, (1977) argue that plant height and density appear to have little or no effect in the selection of oviposition sites for the species Costelytra zelandica (White) and Heteronychus arador (Fabricius). However, Szendrei & Isaacs, (2005), in studies of other species of scarabaeid established that females of Popillia japonica Newman can discriminate between the quality and quantity of artificial shoots, as well as the role of vertical objects on the soil surface. The latter suggests that oviposition is selective and is influenced by cues from plants. According to Régnière et al., (1981) and Allsopp et al., (1992), in laboratory experiments with P. japonica, females were selective in their oviposition in accordance with soil characteristics like texture, moisture and organic matter. Ward & Rogers (2007), demonstrated that the soil moisture level and its interaction with soil type influences selection of oviposition site in some species more than in others. Gaylor & Frankie (1979) established that extreme moisture levels significantly influences the oviposition selection of Phyllophaga crinita (Burmeister). These results show that far from behaving as a group, scarabaeids present species specific oviposition behavior.

The selection of an adequate oviposition site in phytophagous insects is a crucial factor in their survival and the subsequent development of their offspring, because of which it is argued that the female must lay eggs according to a selection-performance relationship.

Considering that the distribution of plants is heterogeneous, the selection of adequate sites is fundamental for the success of offspring (Thompson, 1988). Given the variation in oviposition behavior that occurs in the same species (Thompson & Pellmyr, 1991), natural selection should favor oviposition sites that optimize the survival of offspring (Holland et al., 2004). The degree of host discrimination can be influenced by ecological conditions (Roitberg et al., 1999). This situation is particularly important among insects that feed on limited resources and where the insect, in its immature stages, presents little movement (Suguira et al., 2007). Nevertheless, the selection of oviposition sites also occurs in polyphagous insects, given that in the first or early stages the larvae have a limited capacity to search for food owing to their small size and limited ability to tunnel through the soil (Szendrei & Isaacs, 2005).

Scarabaeoids and other insects tend to present highly aggregated distribution, according to Andersen, (1987) and Brown & Gange, (1990), spatial distribution being one of the intrinsic properties of insect species (Taylor, 1961) and as a result of intra- and inter- species interaction and habitat (Hunter, 2001). This aggregated distribution of scarabaeid larvae is the product of the physical, chemical and environmental characteristics of the soil, including texture, compaction, pH, temperature, moisture, organic matter and the nutritive condition of the soil (Laughlin, 1963; Ridsdill Smith, et al., 1975; Vittum & Tashiro, 1980; King et al., 1981; Régnière et al., 1981; Potter, 1983; Vittum, 1984; Curry, 1987; Villani & Wright, 1990) and probably the selection of oviposition sites by adults, as occurs in species of other orders (Gotthard et al., 2004).

Our objective was to examine how soil type, soil substrates and plant material influence P. herrmanni oviposition site selection. We assessed the attractiveness of plant and soil characteristics to P. herrmanni. females. For visual cues we investigated the response of P. herrmanni oviposition site selection to plants and dummy plants and the response to the quantity of the stimuli.

MATERIALS AND METHODS

Insects: Adult P. herrmanni were collected in the field at the Santa Rosa Experimental Station (39°47’S and 73°14’ W) of the Universidad Austral de Chile. Each insect was sexed according to its external characteristics. The adult insects were kept for 24 h in plastic containers (10 L), without foliage before being released into cages. Some 400 adults, at a sexual rate of 1:1, were released into each cage.

Field oviposition arena: Two cages of white netting (antiafide 16/10 40 mesh, Tenax ®) measuring 22 m3 (6 x 2 x 1.85 m) were installed at the Santa Rosa Experimental Station (39°47’22’’S and 73°14’01’’W) of the Universidad Austral de Chile. The cage floors were covered with black plastic 2mm thick (Filmoamérica, Brazil). Treatments were established by making 80 equidistant holes, with a steel core (10x10cm). Each hole was lined with an empty plastic pot (Total Pack ® 14 Argentine) of 1.4 L, inside of which was a pot of similar texture, size and volume. The pots, filled with each treatment were weighed to ensure uniform weight. Three times a week three pots of each treatment were removed and weighed to establish moisture conditions. Water was added if necessary, to maintain uniform moisture levels for all the treatments

Soil and substratum: The soils used were collected from different locations in Los Lagos and Los Rios Regions. The soils used were: 1) Inceptisol (Huiño Huiño soil series), Aquandic Humaquept: (40°43’S and 72°48’W) 139 m elevation. 2. Ultisol (Huilma, soil series), Andic Palehumults: (40°47’S and 73°16’W) 108 m elevation. 3. Andisol (Puerto Fonk, soil series), Pachic Melanudands: (40°39’S and 72°39’W) 219 m elevation and 4. Andisol (Valdivia, soil series), Duric Hapludands: (39°45’ S 73°14’ W) 13 m elevation (Ciren, 2003). The substrata were river sand and sawdust (> 90 % Nothofagus obliqua (Mirb.) Oerst).

Plants and roots: For the plant treatments in both seasons, we used 3 g of ryegrass seeds Lolium multiflorum Lam. cv. Tama per pot. For the root treatment in the first season, Lolium spp (>90 %) roots were field collected. Each treatment had 1 cm-long roots, which were washed and covered with a layer of soil 1 cm deep. In the second experimental year, roots were obtained differently. The experiment was set up by sowing 3 g of ryegrass cv. Tama 20 days before the beginning of the bioassays. One day before starting the experiment the foliage of plants was cut below soil level and covered with 1 cm of soil.

Covers: The studied soil cover consisted of: 1. chopped oat straw; 2. grated cow manure; 3. sawdust (> 90 % N. obliqua); 4. bare ground, and 5. pasture grass (Lolium perenne L.) and clover (Trifolium repens L.) The pasture had 2 cm height. The soil used in these experiments was from the Valdivian Andisol Series. Cover was applied on the soil forming a uniform 2 cm layer (Figure 1).

Laboratory oviposition arena: Bioassays were carried out in a cage made with a plastic box (Wenco®) of 35 x 24 x 13 cm installed over a base of Aislapol ® 50 mm, density 10 kg mˉ2 (Basf Group, Chile) (external dimensions 50 x 30 x 10 cm of height). Four experimental arenas, each 7.5 cm diameter for oviposition selection, were made at center of each cage. White plastic glasses of 300 mL (Mater, Chile) were used as pots. The soil used was an Andisol from the Valdivia Series, with a humidity of 35 ± 5 % w/w. The treated soil blocks were installed in a cage in a randomized pattern. All arenas were placed in the environmental chamber at 20 ± 2 ºC, 60 ± 5 % r.h. and L16:D8 for 35 to 40 days.

Bioassays

Experiment 1: To determine the oviposition response of P.herrmanni to the density of artificial stalks without roots, 6.5 cm long and 2 mm diameter wooden toothpicks (Marco Polo ® ICB S.A.) were used. The densities were: bare ground (0); 1133.5 (5); 2267 (10)

and 4534 (20) wooden sticks mˉ2 (wooden stick potˉ¹). Each wooden stick was buried 1 cm in the soil. Twenty arenas were established for this experiment.

Experiment 2: To determine the oviposition response of P.herrmanni to the density of ryegrass L. multiflorum Lam. cv. Tama roots, the treatments were sowed 20 days before the beginning of the experiments. The plant foliage was cut below the soil level and covered with 1 cm of soil, 24 h before starting the trial. The treatments were: bare ground (0) g roots; 0.023±0.04 g roots DW /pot-1 equivalent of 0.1g of seeds (23 seeds pot-1); 0.104±0.02 g roots DW /pot-1 equivalent of 0.5 g of seeds (115 seeds pot ˉ¹) and 0.188±0.03 g roots DW /pot-1 equivalent of 1g of seeds (230 seeds pot-1). Twenty arenas were established for this experiment.

Experiment 3: P.herrmanni response to the density of roots 0.188±0.03 g DW potˉ¹ was assessed by evaluating their interaction with dummy plants 4.534 toothpicks mˉ2 at 20 days after sowing. The treatments were: without roots/without foliage; with roots/with foliage; without roots/with foliage and with roots/without foliage. Twenty arenas were established for this experiment.

Assessment of oviposition The number of eggs and larvae found in each soil block was recorded. In the field experiment and laboratory bioassays each soil block was strained (16, 9 and 4 mesh) with a sieve (U.S. Standard Sieve Series, Soiltest Inc.) and handly sorted L1 larvae were collected with forceps and eggs collected with a paintbrush, between 35 and 40 days after introducing the adults.

Statistical analysis The field experiments had a randomized block design in factorial arrangement (5x2), 5 soils or substratum, with and without plants. The experiment with soil covers used the same experimental design, 4 covers, with and without roots. Each treatment had 8 replicates and

was evaluated using an ANOVA test (PROC GLM, SAS Institute), (SAS Institute, 2009). The laboratory experiments evaluated the average number of insects per soil block and then that number was compared to the other treatments estimating oviposition ODD ratio, using logistic regression (PROC Logistic, SAS Institute), (SAS Institute, 2009). The critical value for significance for all these tests was determined at P ≤ 0.05.

RESULTS

Field experiment

Effect of soil type, sawdust, sand and plant presence on selection of oviposition sites Figure 2 shows how soil or substrate type affected the selection of oviposition site averaging two seasons. The difference among soil and substrate types was significant (F=3.74, P<0.0062). Females of P. herrmanni showed preference for sites with plants when these were present (F=20.31, P<0.0001). Oviposition in sand, sawdust and Inceptisol was significantly lower than in ultisol and andisol soils. There was an interaction between plants and season (F=12.97, P<0.0004). Oviposition in season was significantly (F=4.88, <0.028). The sand without plants was not selected in either season by P. herrmanni females as an oviposition substrate, although the presence of plants was a determining factor in the selection. A significantly lower number of eggs were laid in sawdust than in soils in both seasons.

Effect of soil covers and plant roots on selection of oviposition sites by P.herrmanni The results show that in both seasons ground cover affected the oviposition conduct of P. herrmanni females (Fig 3 and 4). The effect of cover was significant in both seasons (F=4.49, P<0.0027 and F=5.30, P<0.0028) for first and second season respectively. In the first season, the main oviposition preference was the treatment with pasture and oat straw, with and without plants roots. In the second season the ground cover with oat straw had a significantly higher level of oviposition. The presence of plants roots significantly increased oviposition in both years compared to cover (F=11.60, P<0.0011 and F=6.45,

P<0.0139). The highest rate of oviposition obtained in the first season among the different treatments was probably due to the location and quantity of roots; in the first year roots were placed as a horizontal layer below the surface, while in the second year roots were obtained from living plants seeded in pots, whose foliage was removed and roots covered with 1cm of soil. There were interactions between ground covers and plant roots in both seasons (F=4.34, P<0.0034 and F=2.82, P<0.0469). The cover of cow manure, sawdust and bare ground without roots was not selected by females for oviposition in the second season, but only bare ground without roots was not select in both seasons.

Laboratory experiment

The use of visual cues by female P.herrmanni to select oviposition sites P. herrmanni females use visual cues to select oviposition sites. When 6.5 cm sticks were used as dummy plants, females only selected the treatment with the highest density of dummy plants, indicating that females also respond to density .There was no oviposition in bare soil nor in soils with low or medium densities of dummy plants (Fig 4). There was a close relationship between insect number and plant dummy density (P<0.05).

Effect of chemical cues on oviposition behavior To measure the effect of chemical cues on oviposition behavior of P. herrmanni females, different biomass of roots were used in each pot. Significant differences in the response to the biomass of roots were determined, with the highest quantity of biomass differing most significantly from the other treatments (P<0.05), (Fig 5). Treatments with bare soil and low density of roots were not selected by P. herrmanni females as oviposition sites.

Importance of visual and chemical cues on the selection of oviposition sites by female P.herrmanni: P. herrmanni responds mainly to chemical and visual cues when selecting an oviposition site, with chemical cues being more significant (Fig 6). Significantly, more eggs were

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found in soils with roots and dummy plants than in soils without roots (P<0.05). Bare soil and soil without roots were not selected as oviposition sites. There was a positive interaction between chemical (roots) and visual (dummy plants) cues.

DISCUSSION

The results obtained confirm that scarabaeids, despite their ample phytophagy and capacity to use organic matter from the soil in their diet in their larval stage, respond in the selection of oviposition site to stimuli in the medium that can act as positive or negative cues.

Live plants, parts of plants or simulated foliage in the form of wooden stakes, have a marked effect on the selection of oviposition sites by adult P. herrmanni females. When the effect of density alone was measured by using 6.5-cm-high wooden sticks, 100% of oviposition occurred in the pots with the maximum density of simulated foliage (20 dummy plants per pot). Using both simulated plants and roots, females did not select soil with only simulated plants, but there was a positive interaction between roots and simulated plants. The results confirm the importance of visual and chemical cues in the colonization of new areas by females of the species. The oviposition of P. herrmanni females can occur in two ways, one in which females emerge from galleries under the ground to mate with males above ground and then return underground in the same gallery from where they emerged. In the second, females abandon these sites in flight and colonize new spaces. The study measured oviposition in the latter case. The response obtained corroborates the importance of the visual response of females to vertical objects in the selection of oviposition sites, which can be considered as a pre-alighting response that can be reinforced with olfactory or alimentary response after alighting. In other species of scarabaeids, such as the case of P. japonica, the authors considered these responses as pre- and post- digging in the soil (Szendrei & Isaacs, 2005). We prefer the terms pre- and post-alighting in the selection of

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oviposition, because we believe there is an attraction at greater distance that is not related directly to digging. When artificial stalks were used as the only stimulus, it was observed that the visual response allowed females to discriminate adequately to the intensity of the cue of vertical objects (Szendrei & Isaacs, 2005). However, when females were exposed to visual cues (artificial stalks) and products produced by roots, the females did not colonize places without roots, but when the two stimuli were used simultaneously (roots and artificial stalks) the response was reinforced. This indicates that the colonizing behaviors of P. herrmanni have a pre-alighting visual response that requires re-enforcing by a post- alighting response to cues provided by plants, which can have a lesser pre-alighting response according to the results obtained. Both responses could be caused by C02 or other gaseous compounds produced by plants, given that adult P. herrmanni do not feed, so that an alimentary component can be ruled out (Durán, 1954). The greater response by this species to visual and chemical components, compared to that of H. elegans, is in line with the colonization of new spaces by P. herrmanni, which can remain infesting the new space for several years. Because of this last aspect, the election of an adequate oviposition site is more important than that of another species that abandon oviposition sites annually.

The information obtained reaffirms the hypothesis put forward by Jaenicke, (1978) about selection-performance in the plant insect-plant relationship, which indicates that there is a strong process of natural selection, so that females choose oviposition sites with plants where the possibility of survival, growth and reproduction of their offspring (here on termed performance) is the most adequate, given that in general the performance of this species is better in the presence of plants (Cisternas & Carrillo published elsewhere).

The reason because an insect such as P. herrmanni, with such an ample phytophagy and which can in certain measure feeds on organic soil matter, chooses its oviposition sites in accordance with the selection-performance hypothesis is due to the small size of the larvae and their limited power of penetration, which seriously limits the capacity to search for food in the case where the oviposition site is not adequate for survival and development in

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the immature stage (Régnière et al., 1981.) The stimuli responsible for this response should necessarily be general, as occurs in this case, these being the presence of plants, plant density and CO2, which could produce a similar response in numerous other species of polyphagous insects that consequently can share the same species of plant. This can produce problems of inter-species competition for the same resource among insects whose larvae live in the soil and could explain the presence of populations with a high predominance of one species in the white grub assemblage. However, another factor could be the cause of assemblages of scarabaeid larvae with the predominance of one species given that studies of P. herrmanni and H. elegans have shown the absence of inter-species competition among larvae of the second and third instar of the two species in high densities, but it is not know what occurs with larvae of the first instar (Rothmann, 1994).

The absence of specific cues in the selection of oviposition sites could explain the rapid colonization of plants introduced to Chile (Eucalyptus sp, Triticum sp, Vaccinium sp), by this and other scarabaeids that feed on the roots of native plants. Nevertheless, this response to general stimuli could explain why this species and others do not always oviposit in accordance with the selection-performance theory in the case of plants, as also occurs with other phytophagous polyphagous insects. They may select legumes because of height and density instead of selecting grasses, although their performance with the latter would be better (Rojas, 1994). This is explained by the role of relative abundance of the host, which is known as patch dynamics hypothesis (Thompson, 1985).

In relation to the role of soil type, sand and sawdust in the selection of oviposition site, soils without plants showed no differences in selection in the first year of the experiment but did in the second, where females preferred ultisol and andisol soil. The preference for this type of soil could be related to the characteristic of this species of penetrating deeply into the soil to oviposit, because of which soil characteristics that offer facility to perforate and form tunnels in the soil could be fundamental. When sand was offered as a substrate, there was a marked interaction with the presence of plants. This could indicate that the

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abrasive effects of sand particles (Hoback & Golick, 2000) on the adult are less important in this species than in H. elegans and that, owing to the greater depth of ovipositing, P. herrmanni could have fewer problems of eggs drying out and in embryogenesis, a process during which scarabaeid eggs need to absorb water (Potter, 1983). The sawdust was the substrate that presented the lowest response with plants and in general was least selected by females, which is in line with the selection of oviposition sites that offer optimal conditions for offspring, given that in studies with Diptera, sawdust has been shown to have negative effects on survival and development in the immature stages (Schmidtmann, 1991).

The effect of different soil covers in the selection of oviposition site by females was analyzed independently. The pasture cover and the manner in which roots were placed were different in the two years. In the first experimental year a layer of roots were placed on the soil and covered by 1 cm of soil and 2 cm of the cover. In the second year, the aerial parts of the plants were cut and the roots covered with a layer of soil and a layer of cover, similar to the first year. In the first year, P. herrmanni preferred to oviposit in soil covered by oat straw and manure and to a lesser degree, by pasture that did not have roots and was cut very short (1cm high). This preference for type of cover could be due to the greater presence of moisture, which is essential for the embryogenesis of the scarabaeid eggs that need to absorb water from the medium (Fresard, 1992). As well, covers like manure can produce C02 and nitrogen products that favor election of oviposition sites (Hadas, et al., 1998). The low response to natural pasture of a low height (1 cm) without roots is notably, indicating that the height of foliage is an extremely important aspect in selection and that the color green as limited importance, confirming the role of vertical objects of a certain height in attracting scarabaeids, as pointed out by Szendrei & Isaacs, (2005). In the second year only oat straw produced a response among covers without roots, which could be due to the transformation processes in the manure that reduce the release of gases, so that its attraction to females is reduced. When roots that remain biologically active were put under the cover below 1 cm thick of soil, females responded especially to the presence of manure and oat straw in interaction with the roots.

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In summary, the results obtained affirm that P.herrmanni responds to general stimuli in the search for an oviposition site and that in this species, the response go chemical and physical (visual) stimuli is greater than in H.elegans (Cisternas & Carrillo published elsewhere). This greater response to the presence of compounds like C02 in this species could be related to the more phytophagous habits of Melolonthinae compared to Rutelinae (Ritcher, 1958) and the distinct colonization of new spaces, as discussed earlier. As to whether this response is related to offspring performance, it is in general terms (plants- absence of plants-plant height), but it is not possible to affirm its power of discrimination between plants that address the larval requirements can be very specific. Consequently, the pressure of selection exercised by plants must be reduced. For that reason preference for knew host plants may be irrelevant , but if the colonization of new hosts (Thompson, 1988), such as has been occurring in Chile , but without abandoning their old host plants.

ACKNOWLEDGEMENTS

We are grateful to Sonia Santana H., Jorge Barría M and Manuel Muñoz, owners of the farms where soil samples were obtained. We wish to thank to the Soil Laboratory personnel at the Universidad Austral de Chile. We also thank Gustavo Valdebenito for his assistance in the field, and Hector Uribe for advice on the statistics.

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Aguilera PA, Cisternas AE, Gerding PM & Norambuena MH (1996) Plagas de praderas. Praderas para Chile. (ed. by NI Ruiz). Impresos Offset Bellavista Ltda. Santiago, Chile, pp. 309-339. Allsopp PG, Klein MG & McCoy EL (1992) Effect of soil moisture and texture on oviposition by the japanese beetle and rose chafer (Coleóptera:Scarabaeidae). Journal of Economic Entomology 85: 2194-2200. Andersen D (1987) Below-ground herbivory in natural communities: A review emphazing fossorial animals. The Quaterly Review of Biology 62: 261-286. Brown VK & Gange AC (1990) Insect Herbivory Below Ground. Advances in Ecological Research 20: 1-58. Carrillo R (1986) Plagas en praderas. Producción de forrajes. (ed. by L Latrille). Universidad Austral de Chile, Facultad de Ciencias Agrarias. Instituto de Producción Animal. Valdivia, Chile, Serie B-ll pp. 76-94. Ciren (2003) Descripciones de suelos, materiales y símbolos. Estudio agrológico X Región. Santiago de Chile, Chile. Publicación CIREN N° 123 Tomo I y II. 412 p. Curry JP (1987) The invertebrate fauna of grassland and its influence on productivity. II. Factors affecting the abundance and composition of the fauna. Grass and Forage Science 42: 197-212. Durán L (1954) La biología de Phytholoema herrmanni Germ. y mención de otros escarabeidos perjudiciales a la agricultura de las provincias australes de Chile. Revista Chilena de Historia Natural 54: 5-20. Durán L (1976) Problemas de la entomología agrícola en Chile Austral. Agro Sur 4: 119- 127. East R & King PD (1977) Effect of botanical composition of pastures on insect pest populations. The New Zealand Entomologist 6: 273-278.

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Fresard ME (1992) Aspectos biológicos de Hylamorpha elegans Burm. y fitofagia de larvas de dos especies de escarabaeidos en plantas de trigo. Tesis Lic. Agr., Facultad de Ciencias Agrarias, Universidad Austral de Chile 145 p. Gaylor MJ & Frankie GW (1979) The relationship of rainfall to adult flight activity; and of soil moisture to oviposition behaviour and egg and first instar survival in Phyllophaga crinita. Environmental Entomology 8: 591-594. Gotthard K, Margraf N & Rahier M (2004) Geographic variation in oviposition choice of a leaf beetle: the relationship between host plant ranking, specificity and motivation. Entomologia Experimentalis et Applicata 110: 217-224. Hadas A, Parkin TB & Stahl PD (1998) Reduced C02 release from descomposing wheat straw under N-limiting conditions: simulation of carbon turn over. European Journal of Soil Science 49:487-494. Holland JN, Buchanan AL & Loubeau R (2004) Oviposition choice and larval survivasl of an obligately pollinating granivorous moth. Evolutioinary Ecology Research 6:607- 618. Hoback WW & Golick DA (2000) Salinity and shade preferences between sympatric tiger beetle species Ecological Entomology 15: 180-187. Hunter MD (2001) Out of sight, out of mind: the impacts of root-feeding insects in natural and managed systems. Agricultural and Forest Entomology 3: 3-9. Jaenicke J (1978) On optimal oviposition behavior in phytophagous insects. Theoretical Population Biology 14: 350-356. King PD, Mercer CF & Meekings JS (1981) Ecology of Black Beetle Heteronychus arator, influence of plant species on larval consumption, utilization and growth. Entomologia Experimentalis et Applicata 29: 109-116. Klein C & Waterhouse D (2000) Distribution and importance of arthropods associated with agriculture and forestry in Chile. ACIAR Monograph N° 68 231 p. Laughlin R (1963) Biology and ecology of the garden chafer Phyllopertha horticola (L.) VIII. Temperature and larval growth. Bulletin Entomological Research 54: 745-759.

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Potter DA (1983) Effect of soil moisture on oviposition water absorption, and survival of southern masked chafer (Coleoptera:Scarabaeidae) eggs. Environmental Entomology 12: 1223-1227. Régnière J, Raab RL & Stinner RE (1981) Popillia japonica: Effect of soil moisture and texture on survival and development of eggs and first instar grubs. Environmental Entomology 10: 654-660. Ridsdill Smith TJ, Porter MR & Furnival AG (1975) Effects of temperature and developmental stage on feeding by larvae of Sericesthis nigrolineata (Coleoptera: Scarabaeidae). Entomologia Experimentalis et Applicata 18: 244-254. Ritcher P (1958) Biology of Scarabaeidae. Annual Review of Entomology 3: 311-334. Rivera M (1904) Biología de dos coleópteros chilenos cuyas larvas atacan al trigo. Revista Chilena de Historia Natural 8:41-254. Roitberg DB, Roberson CI & Tyerman GA (1999) Vive la variance: a funcional oviposition theory for insects herbivores. Entomologia Experimentalis et Applicata 91:187-194. Rojas E (1994) Estudio de las relaciones entre diferentes densidades larvales de escarabaeidos y diferentes especies pratenses. Tesis Lic. Agr., Universidad Austral de Chile, Facultad de Ciencias Agrarias 135 p. Rothmann S (1994) Evaluación de competencia entre larvas de dos escarabaeidos nativos Tesis Lic. Agr., Universidad Austral de Chile, Facultad de Ciencias Agrarias. 64 p. SAS Institute (2009) Version 9.1.3. SAS Institute, Inc, Cary, NC. Schmidtmann ET (1991) Suppressing immature house and stable flies in outdoor hutches with sand, gravel and sawdust bedding. Journal of Dairy Science 74: 3956-3960. Szendrei Z & Isaacs R (2005) Do plant cues influence the oviposition behavior of Japanese beetles? Entomologia Experimentalis et Applicata 117: 165-174. Sugiura S, Yamazaki K & Yamaura Y (2007) Intraspecific competition as a selective pressure on the choice of a oviposition site in a phytophagous insect. Biological Linnean Society 92:641-650. Taylor LR (1961) Aggregation, variance and the mean. Nature 189: 732-735.

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Thompson JN (1985) Within–patch dynamics of life histories, populations and interactions: selection over time in small spaces. The ecology and natural disturbance and patch dynamics. (ed. STA Pickett & PS White) Academic Press, New York, USA, pp. 253- 264. Thompson JN (1988) Evolutionary ecology of the relationship between oviposition preference and performance of offspring in phytophagous insects. Entomologia Experimentalis et Applicata 47: 3-14. Thompson JN & Pellmyr O (1991) Evolution of oviposition behaviour and host preference in Lepidoptera. Annual Review of Entomology 36: 69-89. Villani MG & Wright RJ (1990) Environmental influences on soil macroarthropod behavior in agricultural systems. Annual Review of Entomology 35: 249-269. Vittum PJ & Tashiro H (1980) Effect of soil pH on survival of Japanese beetle and European chafer larvae. Journal of Economic Entomology 73: 577-579. Vittum PJ (1984) Effect of lime applications on Japanese beetle (Coleoptera: Scarabaeidae) grub populations in Massachussetts soil. Journal of Economic Entomology 77: 687- 690. Ward A & Rogers J (2007) Oviposition response of scarabaeids: does ‘mother know best’ about rainfall variability and soil moisture? Physiological Entomology 32: 357-366.

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Table 1. Soil texture of the tested soils

Clay Silt Sand Soil Type Texture % Andisol 18.6 63.9 17.5 Silt loam Inceptisol 27.0 64.3 8.7 Silt loam Ultisol 34.9 54.3 10.8 Clay silt loam

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Figure 1 Roots location in the soil cover experiments.

Cover cover Soil soil Soil

roots

soil

First season Second season

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Figure 2 Oviposition preference of P. herrmanni for different soil types, sand, sawdust and plants, average for two seasons.

120 A A B 100

80 B 60 B 40 B

20 Average number/ of insects pot Average

0 Sand Ultisol Inceptisol Andisol Sawdust

Without plant With plant

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Figure 3 Effect of soil covers and roots on the oviposition behavior of P. herrmann: first season.

250 A

200

150 B

100 B B AB

50 Average number/ of insects pot Average

0 Bare ground Sawdust Manure Oat straw Pasture

Without roots With roots

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Figure 4 Effect of soil covers and roots on the oviposition site selection of P. herrmanni: Second season.

80 A 70

30 B 20 B B

10 Average number/ of insects pot Average

0 Bare ground Sawdust Manure Oat straw

Without roots With roots

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Figure 5 Effect of different densities of dummy plants on P. herrmanni oviposition site selection.

10 9 8 7 6 5 4 3 2

Average number/ of insects pot Average 1 0 0 5 10 20 Dummy plant density (number / pot)

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Figure 6 Effect of the quantity of roots on the oviposition site selection behavior of P. herrmanni.

5 4,5 4 3,5 3 2,5 2 1,5 1

Average number/ of insects pot Average 0,5 0 0 0.1 0,5 1 Roots density ( g of seeds / pot)

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Figure 7 Effect of visual and chemical cues on the selection of oviposition sites by P. herrmanni females. H. elegans. WR: Without roots; WF: Without foliage; R: Roots; F: Foliage.

2 Average number/ of insects pot Average 1

0 WR/WF WR/F R/WF R/F

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8. EFECTO DEL TIPO DE SUELO Y LAS RAICES SOBRE EL CRECIMIENTO Y DESARROLLO DE LAS LARVAS. Hylamorpha elegans (Burm.) (COLEOPTERA: SCARABAEIDAE: RUTELINAE).

Este artículo puede ser consultado bajo el título:

Cisternas, A. E.; Carrillo, Ll. R. y Millas, P. GROWTH AND DEVELOPMENT OF Hylamorpha elegans (Burm.) RUTELINAE (COLEOPTERA: SCARABAEIDAE) IN DIFFERENT SOIL TYPES WITH AND WITHOUT ROOTS. Environmental Entomology (Submitted).

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GROWTH AND DEVELOPMENT OF Hylamorpha elegans (Burmeister) RUTELINAE (COLEOPTERA: SCARABAEIDAE) IN DIFFERENT SOIL TYPES, WITH AND WITHOUT ROOTS

Ernesto Cisternas1*; Roberto Carrillo2 and Paz Millas1 1 Graduate School, Faculty of Agricultural Sciences, Universidad Austral de Chile 2 Laboratory of Entomology, Institute of Production and Plant Protection, Universidad Austral de Chile, P.O. BOX 567 Valdivia, Chile * [email protected]

ABSTRACT

The growth and development of Hylamorpha elegans (Burm.) (Coleoptera: Scarabaeidae: Rutelinae) larvae were studied in laboratory experiments in three soil types, with and without roots. Larval survival, growth and development were affected in different ways by soil types. Survival and growth levels of H. elegans were higher in Inceptisol soil than in Andisol and Ultisol soils. There was no relationship between larval weight and the development beyond the larval stage. Larvae were only able to reach the pupal and adult stages in soils with living roots.

Key words: Scarabaeidae, Rutelinae, Hylamorpha elegans, larval growth, survival

INTRODUCTION

The effects of live roots and soil type on the development and growth of larvae of Hylamorpha elegans (Burm.) has not been studied and remains largely unknown. Larvae of H. elegans represent the most common and widely distributed member of the assemblage of scarabaeid larvae that feed on roots of the different species that compose pastures, as well

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as other arbustive and arboreal species of economic importance in southern Chile (Durán 1952; Durán 1954; Carrillo 1986; Artigas 1994; Aguilera et al. 1996; Klein and Waterhouse 2000). H. elegans is endemic to southern cone of South America, found from Valparaiso Region to Los Lagos Region (Prado 1991; Artigas 1994; Klein and Waterhouse 2000). The species has an annual cycle with periods of variable seasonal flight that occur in the Los Lagos Region between week 47 and 8 (Cisternas, unpublished).

The survival, growth and development of larvae in soil can be affected by biotic factors such as diseases caused by bacteria, fungi, protozoa, viruses, nematodes and other agents (Glare and Jackson 1992), as well as biotic factors (Kard et al. 1988; Villani and Wright 1988). The quality of the food consumed during the larval period also influences insect morphology, growth, reproduction and survival (Logan et al. 2001; Logan and Kettle 2002; Karino et al. 2004). Larvae bred under a regime of poor nutrition have a shorter larval stage and pupate prematurely, leading to an early emergence of small adults (Moczek 1998; Shafiei et al. 2001). However, adverse nutritional conditions in other species translate into an extension of the larval stage, so that the insect does not reach the specific size threshold for its metamorphosis (Esperk et al. 2007). The presence or absence of roots is an important factor for the adequate development of some species (Farrell 1972; Ridsdill Smith 1975; Berry and Potter 1995; Logan and Kettle 2002). Plant exudates do not produce variations in the larval weight of the third instar of H. elegans (Rojas 2005). The majority of insects that feed on roots are considered polyphagous herbivores. Nevertheless, some exhibit preferences for some species, confirming the existence of favorable and unfavorable plant species (East and King 1977; King et al. 1981; Prestidge et al. 1985). Physical and chemical characteristics of soils, such as OM, pH, humidity, temperature, structure, porosity and aeration also affect invertebrates (Régniére et al. 1981; Potter and Gordon 1984; Vitum 1984; Curry 1987; Brown and Gange 1990). Artificial fertilizers have little or no effect on growth and survival of Costelytra zealandica (White) (Prestidge et al. 1985). Feeding during the larval stage can have important effects on the development of scarabaeid populations, especially with those species that do not feed as adults. Fecundity in many

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insects is related to the pupal and adult weight, because of which it is closely related to larval feeding, especially in species that do not feed as adults (Logan et al. 2001; Calvo and Molina 2005; O’Neill et al. 2008).

In his book on scarabaeids, Ritcher (1958) accepted the proposal of De Fluiter and classified larvae according to their feeding habits, pointing out that larvae that only feed on dead organic matter belong to the Cetoniinae subfamily. Larvae that consume organic matter, but in its absence consume roots, belong to the Rutelinae or Dynastinae subfamilies, while larvae that feed mainly on plant roots belong to the Melolonthinae.

The objectives of this study were to establish the effect of the three most important soil types of southern Chile, with and without roots, on the survival, development and growth of the larval stage of H. elegans and the formation of the pupal and adult stages.

MATERIALS AND METHODS

Insects. Adult H. elegans were collected in the field at the Universidad Austral de Chile’s Santa Rosa Experimental Station (39°47’S and 73°14’W). Each insect was sexed according to its external characteristics (Cisternas 1986). The adult insects were kept for 10 days in plastic containers (10 L), with soil type Andisol serie Valdivia and foliage of Nothofagus obliqua (Mirb.) Oerst. During the rearing period, the foliage of N. obliqua in the containers was changed three times a week. The eggs obtained every 48 hours were moved to terrariums with earth and kept at a constant temperature of 18 ± 2ºC. Beginning on day 15 of incubation, the terrariums were checked every 24 hours to obtain neonate larvae (L1). Each of the L1 larvae obtained in the laboratory was carefully handled and selected for its physical condition and its capacity to enter the soil. Once its weight was registered with an analytical scale (Precisa 100A-300M), the selected larvae were placed individually in polypropylene 100 mL pots and closed with a cap with small holes. After 24 h the L1 larvae that had not buried themselves by their own means were replaced. The treatments

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were maintained under constant laboratory breeding conditions, with a temperature of 18 ± 2 ºC, relative humidity of 50 ± 10% and a photoperiod (L:D 0:24). Every 15 d the live larvae were weighed and the larval stage and mortality were registered up to 300 days. The relative growth rate (RGR) was also determined (RGR) = (LN final weight (mg) – LN initial weight (mg)) / Nº of days between observations (Adams and Van Emden 1972). Plants of Triticum aestivum cv. Kumpa, in number of four plants per pot, provided living roots. These were seeded four days before the beginning of the experiment and before each bi-weekly evaluation. Soil compacting in the pots varied between 1.25 and 1.50 kg cmˉ2, determined with a manual penetrometer (Pocket penetrometer CL-700 / SOILTEST INC. Chicago USA).

Soils

The three soil types used were collected in different localities in the Los Lagos and Los Ríos Regions. Soil 1. Inceptisol (Huiño Huiño, soil series), Aquandic Humaquept: (40°43’S and 72°48’W) 139 m.a.s.l. Soil 2. Ultisol (Huilma, soil series), Andic Palehumults: (40°47’S and 73°16’W) 108 m.a.s.l. Soil 3. Andisol (Puerto Fonk, soil series), Pachic Melanudands: (40°39’S and 72°39’W) 219 m.a.s.l. (CIREN, 2003). Soil humidity was maintained at 35 ± 5 % wt/wt. When the pots lost more than 10% of original weight, the weight was restored with distilled water.

Chemical analysis of the soil, POM and basal respiration

Each soil was chemically analyzed at the Soils Laboratory at the Universidad Austral de Chile. The soils used were characterized by high organic matter content, low pH and low available phosphorus, together with high K content and high aluminum saturation (Table 1).

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The organic matter in each soil was divided according to the size of particulate organic matter (POM) and analyzed to establish the percentage of matter larger than 53 µm. To do this, 10 g of soil were sieved to 2 mm, dried for 48 hours at 30 ºC and dispersed in a solution of 30 mL of sodium hexametaphosphate (5 g Lˉ¹) agitated for 15 hours in a reciprocal agitator at 140 rpm. The dispersed samples were separated with a 53 µm sieve in several washings, until the rinsing water was clear. The material retained in the sieve and the fraction that passed through were dried at 45ºC for 48 hours. Subsequently, C and N were determined (Cambardella and Elliot 1992). (Table 2).

Basal soil respiration was determined by measuring released CO2, using the Isermeyer method. To do this, 10 mg samples of dried fine soil were humidified at 50% gravimetric humidity. The soil was enveloped in a gauze that was suspended through threads within a 1

L glass container with 25 mL of baryta water (7.17 g Ba (OH2) · 8 H2O + 1 g BaCl2, dissolved in a liter of distilled water. The glass flask was covered hermetically and parallel to this a control without soil was prepared. After 24 h of incubation, the barium hydroxide that did not react with CO2, was tittered with HCL (0.1 N), adding some drops of phenolphthalein (Steubing et al. 2002). The soils employed in the research presented high basal respiration, which concurred with the values for volcanic soils as indicated by Zagal, et al. (2002). Inceptisol soil presented a markedly higher level of basal respiration, in the measurement taken at 24 h , it was at least 33% higher than the levels of the other two soil types (Table 3).

Data analysis

The experimental design was a completely random factorial arrangement (3x2), three soil types and two conditions, with and without roots. Each treatment had 60 replications. An analysis of variance (ANOVA) with the PROC GML procedure in SAS (SAS Institute (2009) was used for analysis of the date where the insect nested in the soil / treatment

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interaction was considered as an experimental error to partially remove the correlation that exists upon repeatedly weighing or measuring the weight of an insect over time.

RESULTS

Survival of larvae of Hylamorpha elegans

Survival in the larval stage of H. elegans is presented in (Fig. 1). In general, an increase in survival was observed with the presence of roots, with the exception of Inceptisol under the study conditions. In Inceptisol soil without roots H. elegans is able to grow and develop for a period of 300 d while in the same soil with roots it survives for 270 d. In Andisol without roots the larvae only survive for 15 d and in the same soil with roots for 270 d. In Ultisol without roots larvae survived 90 d and with roots 255 d . In considering the different periods, it was found that at 75 d , 25% of the evaluation period, that the highest survival rate of H. elegans was 53.3% in Inceptisol without roots and the lowest rate, 28.3%, was in the same soil with roots. The survival rate in Andisol soil without roots was 0% and in the same soil with roots it was 15 %. In Ultisol without roots, survival was 3.3%, while in the same soil with roots it was 43.3%. Survival was 0% in the Andisol and Ultisol soils at 150 d at 50% of the duration of the evaluation period. H. elegans did not survive more than 15 and 90 d respectively in the Andisol and Ultisol, in contrast to the behavior of the larvae bred in Inceptisol soil without roots, which survived to 50% of the study period. Survival in Ultisol, Inceptisol and Indisol soils with roots was 15, 28.3 and 38.3% respectively. At 225 d , 75% of the evaluation period, the highest survival rate of H. elegans was 28.3% in Inceptisol soil without roots. In Andisol, Ultisol and Inceptisol soils with roots, the survival rates were 3.3, 5 and 8.3%, respectively.

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Growth of H. elegans larvae

Relative growth rate

The relative growth rate (RGR) of H.elegans larvae according to soil type can be seen in Figs. 2, 3 and 4. In the Inceptisol type soils the larvae have similar RGR in the presence or absence of roots. However, in the Andisol soil the larvae without roots did not survive and those with roots showed a similar RGR to that of larvae bred in Inceptisol with roots. In the Ultisol the RGR of larvae without roots, between 45 and 90 d was different from the RGR of larvae bred in the same soil with roots (P<0.05). The larvae in the three soil types bred with roots presented similar RGR.

Larval growth per instar

The effect of the different soils, with and without roots, on the larval growth was estimated by bi-weekly instar weight. First instar larvae bred in the three soils presented differences among them (P<0.05) and growth in the presence of roots was different from that of larvae bred without roots (P<0.05). The soil-roots interaction was significant (P<0.05). The second instar larvae did not show differences among the three soil types, with and without roots. The third instar did not show differences among the three soil types in terms of their growth, but did show differences when bred with roots (P<0.05) (Table 5).

Larval growth dynamic

The larvae in Inceptisol soil, with and without roots (Fig. 5) maintained continuous growth for 180 d. However, the subsequent decrease in average larval weight was different in the two situations. Among the larvae bred in Inceptisol soil with roots, there was a continuous reduction of average weight until day 270, while the larvae bred in the same soil without roots maintained their average weight until day 300. Both larval groups completed their

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growth with similar weights (451.2 mg and 446.7 mg) with and without roots, respectively. With Andisol and Ultisol soils (Figs. 6 and 7), growth curves are only presented for the treatments with roots since the larvae in the treatments without roots did not survive. In these two soils, as with the Inceptisol with roots, maximum average larval growth was reached between 165 and 180 d , after which there was a continuous reduction in weight from between 255 and 270 d . The larvae bred in Inceptisol and Ultisol soils with roots completed their growth with similar average weights of 451.2 mg and 442.9 mg, respectively, while those bred in Andisol soil with roots had a lower weight of 322.6 mg. Average weights at the end of L3 larval growth were different for larvae bred in soil with roots (537.70 mg) and without roots (425.00 mg). Average weight of larvae bred in Utisol with roots was 431.90 mg and those bred in Andisol soil presented a lower average weight of 386.50 mg (Table 6).

Develop of larvae and formation of the pupal and adult stages

The larvae of H. elegans in Inceptisol, Andisol and Ultisol soils with roots reached the pupal stage at 240, 225 and 210 d respectively (Figs. 8, 9 and 10) and the adult stage at 255, 240 and 255 d respectively. In the three soil types with roots, the L3 stage had a duration of 210 days, the L2 stage between 45 and 75 d and the L1 stage between 30 and 45 d . In the same three types of soils without roots larval development was not completed. In Inceptisol and Ultisol soils the development of the L1 instar was completed between 60 and 75 d . The L2 instar was only completed in the Inceptisol at 90 d . Larval development was different according to the soil type and the presence or absence of roots (P<0,05).

DISCUSSION

The first larval stages L1 and L2 of H. elegans were affected by the soil type and by the absence of roots. In general, larvae survived better and gained more weight when they feed on live roots. This is similar to what was indicated by Logan and Kettle (2002) for the

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species Dermolepida albohirtum (Waterhouse) and by Ridsdill Smith (1975) for Anisoplia austriaca (Herbst.), Popillia japonica Newman, Costelytra zealandica (White) and Sericesthis geminata (Boisduval), the exception being Rhopacea morbillosa Blackburm.

The Inceptisol soil type presented the best condition for the growth, development and survival of larvae, independent of the presence of roots. The higher quality of this soil favored the biological potential of the larvae and was not associated with the quality of organic matter, measured on the bases of the size of particulate matter (Table 2). This is surprising given that Millas (2010), determined that an increase in the labile fraction of OM of the soil constitutes a resource that favors the larval development of scarabaeids, given that it has been demonstrated in other species of scarabaeids that can be consumed selectively from the organic matter fraction of the soil (Mc Quilan and Webb, 1994). However, that case dealt with the same type of soil, while the current work deals with different soil types. On the other hand, basal respiratory activity was 33% higher in Inceptisol soil (Table 3) owing to a higher level of microorganism activity in the soil. This leads to the conclusion that the direct consumption of microorganisms could be more important for the development of H. elegans larvae than the consumption of organic matter. As there is a close relationship between microorganisms and particulate organic matter, the difference observed in this case is because other factors in the soil could affect the type and quantity of microorganisms, such as aluminum saturation (Kanazawa et al. 2005), which was lower in Andisol soils. An important difference among the soils is aluminum saturation and exchangeable aluminum (Table 1). Despite the good growth, development and regular survival of H. elegans larvae in Inceptisol type soils, they can only reach the pupal and adult stages if there were roots in the soil. In Andisol and Ultisol type soils, the development, growth and survival were lower in the absence of roots and H. elegans could not reach the pupal and adult stages in these soils, suggesting that labile organic matter and microorganisms did not provide all the nutritive requirements of H. elegans, while larvae of other insects do not complete their development in the absence of certain amino acids, lipids, sterols, polyunsaturated fatty acids or vitamins (Nation 2002). The fact that the

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larvae reached a similar weight in Inceptisol soil, with and without roots, while they did not pupate without roots, suggests that because the larvae reached a certain threshold of size and weight, neurosecretory cells of the brain sent a message to the protothoraxic glands (Mirth and Riddiford 2007) but the insect probably does not have the necessary sterols to form ecdysteroids to molt and development pupal tissue or that juvenile hormonal levels remain abnormally high to produce the change to the pupal stage.

The greater fitness of larvae reared in Inceptisol soils contrasts with the low populations of larvae found in this type of soil, probably owing to the catastrophic effects of periodic flooding during the winter. These catastrophic effects have been reported among other insect groups (Plum 2005), producing important reductions in the density of insect populations or their extinction. The reduced populations in that are present in Inceptisol soils are individual immigrants that originate from source populations and move to sink populations, avoiding complete extinction (Hanski 1998). In this manner the different populations of scarabaeid larvae in Inceptisol soil correspond to metapopulations, in which the extinction of some populations is a recurring phenomenon. The results indicate that if catastrophic events (flooding) in Inceptisol type soils are reduced owing to drainage, heavy increases in the populations of this and other scarabaeids could occur, possibly reaching plague levels. However attending to the complexity of the interactions between animal populations it is difficult to predict accurate what may occur with the size of the H elegans larva population.

Larval feeding can have important implications in the development of scarabaeid populations. It is clear through this study that H. elegans responds to soil type and the presence of roots. It will be important in future research to determine the effect of different plants on the development of the population, as pointed out by Farrell (1972) and East and King (1977) about populations of C. zealandica, which increase much less under Trifolium spp. than under grasses, the population being suppressed under species such as s Lotus pedunculatus Cav., Medicago sativa L. and Phalaris tuberosa L. In contrast, the population

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of H. arator increases more under grasses than under legumes, reducing the former the larval growth, survival and adult fecundity (King et al. 1981). Larval feeding and nutrition influence larval survival and growth, and adult size, which in turn affect the egg-laying potential, which can be an important factor in population dynamics.

H. elegans is able to feed in part on organic matter present in the soil, which concurs with its ruteline condition (Ritcher 1958), nonetheless, it requires compounds present in roots to complete its development.

ACKNOWLEDGEMENTS

We wish to thank the Soil Laboratory personnel at the Universidad Austral de Chile and Gustavo Valdebenito for his assistance in the field and Leticia Silvestre for her assistance in the laboratory. We are grateful to Sonia Santana H., Jorge Barría M and Manuel Muñoz, owners of the farms where soil samples were obtained. We would also like to thank Hector Uribe for advice on statistics.

REFERENCES CITED

Adams, J., and H. Van Emdem. 1972. The biological properties of aphids and their host plant relationships. pp. 44-47. In: Van Emdem, H. (ed.), Aphid Technology. Academic Press, London. Aguilera, A., E. Cisternas, M. Gerding, and H. Norambuena. 1996. Plagas de las Praderas, pp. 309-339. In I. Ruiz (ed.), Praderas para Chile. Instituto de Investigaciones Agropecuarias, Santiago, Chile Artigas, J. 1994. Entomología económica: Insectos de interés agrícola, forestal, médico y veterinario. Universidad de Concepción, Concepción, Chile. Berry, A. C., and A D. Potter. 1995. Feeding by Japanese Beetle and Southern Masked Chafer Grubs on Lawn Weed. Crop Sci. 35: 1681-1684

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Brown, V.K., and A.C. Gange. 1990. Insect Herbivory Below Ground. Advances in Ecol. Res. 20: 1-58. Calvo, D., and J.M. Molina. 2004. Utilization of blueberry by the lappet moth, Streblote panda. Hübner (Lepidoptera:Lasiocampidae): survival, development and larval performance. J. Econ. Entomol. 97: 957-963. Cambardella, C., and E. Elliot. 1992. Particulate soil organic-matter changes across a grassland cultivation sequence. Soil Sci. Soc. Am. J. 56: 777-783. Carrillo, J. R. 1986. Plagas en Praderas. pp 76-94. In: L. Latrille (ed.), Producción de forrajes. Facultad de Ciencias Agrarias. Instituto de Producción Animal. Universidad Austral de Chile. Valdivia. (CIREN) Centro de Información de Recursos Naturales. 2003. Descripciones de suelos, materiales y símbolos. Estudio agrológico X Región. CIREN N° 123 Tomo I y II, Santiago, Chile. Cisternas, A. E. 1986. Descripción de los estados preimaginales de escarabaeidos en praderas antropogénicas de la zona sur de Chile. Tesis Lic. Agr., Universidad Austral de Chile, Valdivia. Curry, J.P. 1987. The invertebrate fauna of grassland and its influence on productivity. II. Factors affecting the abundance and composition of the fauna. Grass For. Sci. 42: 197-212. Durán, L. 1952. Aspectos ecológicos de la biología del San Juan Verde, Hylamorpha elegans (Burm.) y mención de las demás especies de escarabeidos perjudiciales en Cautín. Agr. Tec. 12: 24-36. Durán, L. 1954. La biología de Phytholoema herrmanni Germ. y mención de otros escarabeidos perjudiciales a la agricultura de las provincias australes de Chile. Rev. Chilena Hist. Nat. 54: 5-20. East, R., and P. D. King. 1977. Effects of botanical composition of pastures on insect pest populations. The New Zealand Entomologist 6: 273-278. Esperk, T., T. Tammaru, and S. Nylin. 2007. Intraspecific variability in number of larval instars in insects. J. Econ. Entomol. 100:627-645.

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Farrell, J.A.K. 1972. Plant resistence to the grass grub, Costelytra zealandica (White) (Col.,Scarabaeidae). I. Resistence in pasture legumes. NZ J. Agr. Res.15: 904-908. Glare.T.R. and T.A.Jackson. 1992. Use of pathogens in scarab pest management. Intercept. Andover, Hampshire UK. Hanski, I. 1998. Metapopulation dynamics. Nature 396: 41-49. Kard, B.M.,F.P. Hain, and W.M. Brooks. 1988. Field suppression of three white grub species (Coleoptera: Scarabaeidae) by entomogenous nematodes Steinernema feltiae and Heterorhabditis heliothidis. J. Econ. Entomol. 81:1033 – 1039. Kanazawa, S., T.T.T. Chau, and M. Shintaro. 2005. Identification and characterization of high acid tolerant and aluminum resistant yeast isolated from tea soils. Soil Sci. Plant Nutr. 51: 671-674. Karino, K., N. Seki, and M. Chiba. 2004. Larval nutritional environment determines adult size in Japanese horned beetles Allomyrina dichotoma. Ecol. Res. 19: 663-668. King, P.D., C.F. Mercer, and J.S. Meekings. 1981. Ecology of black beetle Heteronychus arator, influence plant species on larval consumption, utilization and growth. Entomol. Exp.Appl. 29: 109-116. Klein, C., and D. F. Waterhouse. 2000. Distribution and importance of arthropods associated with agriculture and forestry in Chile. ACIAR Monograph N° 68. Canberra, Australia. Logan, P.D., P.G. Allsopp, and M.P. Zalucki. 2001. Effect of body size on fecundity of Childers canegrub, Antitrogus parvulus Britton (Coleoptera: Scarabaeidae). Aust. J. Entomol. 40: 365-370. Logan, P.D., and C.G. Kettle. 2002. Effect of food and larval density on survival and growth of early instar greyback cane grub, Dermolepida albohirtum (Waterhouse) (Coleoptera: Scarabaeidae). Aust. J. Entomol. 41: 252-261. McQuillan, P.B., and W.R. Web. 1994. Selective soil organic matter consumption by larvae of Adoryphorus couloni (Burmesiter) (Coleoptera: Scarabaeidae).

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J. Aust. Entomol. Soc. 33: 49-50. Millas, P. 2010. Actividad alimenticia de Hylamorpha elegans (Burm.) sobre la materia orgánica particulada y la biomasa microbiana del suelo. Tesis Doctoral, Universidad Austral de Chile, Valdivia. Mirth, C.R., and L.M. Riddiford. 2007. Size assessment and growth control, how adultsize is determined in insects. BioEssays. 29:344-355. Moczek, A.P. 1998. Horn polyphenism in the beetle Onthophagus taurus; larval diet quality and plasticity in parental investment determine adult body size and male horn morphology. Behav. Ecol. 9:636-641. Nation, L. J. 2002. Insect physiology and biochemistry. CRC Press, Boca de Ratón, USA. O’Neill, B.F.,A.R. Zageri, M.R. De Lucia, and M.R. Berembaum. 2008. Longevity and fecundity of japanese beetle (Popillia japonica) on foliage grown under elevated carbon dioxide. Environ. Entomol. 37:601-607. Plum, N. 2005. Terrestrial invertebrates in flooded grassland: a literature review. Wetlands 25:721-737. Potter, D.A., and F.C. Gordon. 1984. Susceptibility of Cyclocephala immaculate (Coleoptera: Scarabaeidae) eggs and immatures to heat and drought in turf grass. Environ. Entomol. 13: 794-799.

Prado, E. 1991. Artrópodos y sus enemigos naturales asociados a plantas cultivadas en Chile. Instituto de Investigaciones Agropecuarias, Bol. Tec. Nº 169. Santiago, Chile. Prestidge, R.A., S. Van der Zijpp, and D. Badan. 1985. Effects of plant species and fertilizers on grass grub larvae, Costelytra zealandica. NZ J. Agr. Res. 28: 409- 417. Régnière, J., R.L. Raab, and R.E. Stinner. 1981. Popilia japonica: Effect of soil moisture and texture on survival and development of eggs and first instar grubs. Environ. Entomol. 10: 654-660.

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Ritcher, P. 1958. Biology of Scarabaeidae. Ann. Rev. Entomol. 3: 31-334. Ridsdill Smith, T. J. 1975. Selection of living grass roots in soil by larvae of Sericesthis nigrolineata (Coleoptera: Scarabaeidae). Entomol. Exp. Appl.18:75-86. Rojas, P. E. 2005. Respuesta de larvas de escarabaeidos a exudados radicales solubles en agua, raíces de distintas especies forrajeras y una enmienda orgánica al suelo. Tesis M.S., Universidad Austral de Chile, Valdivia. SAS Institute, 2009. Version 9.1.3. SAS Institute, Inc, Cary, NC. Shafiei, M., A. P. Moczec, and F. H. Nijhout. 2001. Food availability controls the onset of metamorphosis in the dung beetle Onthophagus Taurus (Coleoptera: Scarabaeidae). Physiol. Entomol. 26: 173-180. Steubing, L., R. Godoy, and M. Alberdi. 2002. Métodos de ecología vegetal. Editorial Universitaria, Santiago. Chile. Villani, M.G., and R.J. Wright. 1988. Entomogenus nematodes as biological control agents of European chafer and Japanese beetle (Coleoptera : Scarabaeidae) larvae infesting turfgrass J. Econ. Entomol. 81: 484-487. Vittum, P.J. 1984. Effect of lime application on Japanese beetle (Coleoptera : Scarabaeidae) grub population in Massachusetts soils. J. Econ. Entomol. 77: 687- 690. Zagal, E., N. Rodriguez, I. Vidal, and L. Quezada. 2002. Actividad microbiana en un suelo de origen volcánico bajo diferentes rotaciones de cultivos. Agr. Tec. 62: 297:309.

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Table 1. Chemical characteristics of the soils used

Parameters Inceptisol Andisol Ultisol

pH in water (1:2,5) 5.7 5.5 5.6

pH CaCl2 (1:2,5) 5.1 4.7 4.8 Organic matter (%) 15.2 22.2 16.6 (ppm N-

Mineral nitrogen NO3) 25.2 28 32.2 Available (ppm P- phosphorous Olsen) 2 5.3 8.3 Exchangeable potassium (cmol+kg-1) 0.33 0.58 0.74 Exchangeable sodium (cmol+kg-1) 0.12 0.12 0.18 Exchangeable calcium (cmol+kg-1) 2.65 2.56 4.64 Exchangeable magnesium (cmol+kg-1) 1.03 0.66 1.14 Exchangeable bases (cmol+kg-1) 4.14 3.93 6.71 Exchangeable aluminum (cmol+kg-1) 0.07 0.68 0.84 CICE (cmol+kg-1) 4.21 4.61 7.55 Al saturation (%) 1.7 14.8 11.1

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Table 2. Particulate matter content of organic matter (POM) greater than 53 µm in the studied soils

Soil type POM > 53 µm (%) Recovery (%) Inceptisol 5.82 ± 0.06 b 97.36 Andisol 6.56 ± 0.06 a 98.03 Ultisol 6.56 ± 0.19 a 94.80

Data from the same column with different letters are significantly different at P< 0.05 as tested with LSD Multiple Range test.

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Table 3. Basal respiration of the soils (CO2 mg. h-1.kg dry soil-1)

Parameter Hours Inceptisol Andisol Ultisol

24 0.36 a 0.24 b 0.23 b Respiration 48 0.26 a 0.19 b 0.13 b

Data from the same row with different letters are significantly different at P< 0.05 as tested with LSD Multiple Range test.

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Table 4. Factorial analysis of variance (ANOVA) with repeated measurements for larval live weight per instar of H. elegans

Instar Source Df SS F P Soil 2 2467.16 57.03 < 0.0001 I Roots 1 31.83 1.47 0.2258 Soil x roots 2 470.51 10.88 < 0.0001 Soil 2 2940.13 0.82 0.4438 II Roots 1 10397.36 5.79 0.0179 Soil x roots 2 8838.02 4.93 0.0288 Soil 2 341134.83 2.36 0.1016 III Roots 1 2197274.69 30.34 < 0.0001 Soil x roots 2

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Table 5. Average weight per larval stage of H. elegans in three soil types, with and without roots

Soil Inceptisol Andisol Ultisol Roots Without With Without With Without With Instar Mean ± SD Mean ± SD Mean ± SD L1 21.99 ± 13.39 19.69 ± 6.62 11.17 ± 3.20 17.29 ± 4.38 17.01 ± 4.11 22.52 ± 4.83 L2 89.86 ± 20.37 90.35 ± 27.98 67.13 ± 15.14 43.97 ± 5.78 98.36 ± 19.06 L3 422.00 ± 56.74 537.70 ± 113.70 386.50 ± 80.35 431.90 ± 69.83

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Fig. 1. Cumulative percentage survival of H. elegans larvae bred in three soils, with and without wheat roots. (I: Inceptisol; IR: Inceptisol roots; A: Andisol; AR: Andisol roots; U: Unltisol; UR: Ultisol roots).

100 90 80 70 60

50 40 30 Cumulative Cumulative % Survival 20 10

15 30 45 60 75 90

105 120 135 150 165 180 195 210 225 240 255 270 285 300 315 Days

I IR A AR U UR

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Fig. 2. Relative growth rates (RGR) of H. elegans larvae in Inceptisol type soil, with and without wheat roots.

7,0

day

day 6,0 / /

/ mg

mg 5,0

/ / /

mg mg 4,0

3,0 larvae

larvae of

of 2,0 . . .

1,0

R.G.RR.G.R 0,0 15 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240 255 270 285 300 Days

No roots Roots

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Fig. 3. Relative growth rates (RGR) of H. elegans larvae in Andisol type soil, with and without wheat roots.

7,0

day day / /

/ 6,0 mg

mg 5,0 / /

/ mg mg 4,0

3,0 larvae

larvae of

of 2,0 . . .

1,0

R.G.RR.G.R 0,0 15 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240 255 270 285 300 Days

No roots Roots

141

Fig. 4. Relative growth rates (RGR) of H. elegans larvae in Ultisol type soil, with and without wheat roots.

7,0

day day / / / / 6,0

mg 5,0 / /

/ mg mg 4,0

3,0

larvae larvae

2,0

of of

. . . 1,0

R.G.R R.G.R 0,0 15 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240 255 270 285 300 Days

No roots Roots

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Fig. 5. Dynamic of the growth of H. elegans per instar in a Inceptisol soil, with and without wheat roots.

900 )

) 800 mg

mg 700

( ( 600 500

weight weight 400

300 Mean Mean Mean 200

100

15 30 45 60 75 90

105 120 135 150 165 180 195 210 225 240 255 270 285 300 Days

L1 L2 L3 L1R L2R L3R

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Fig. 6. Dynamic of the growth of H. elegans per instar in an Andisol soil, with and without wheat roots.

900

) ) 800

mg 700

( ( 600

500 weight weight 400

300

Mean Mean Mean Mean 200 100 0

15 30 45 60 75 90

105 120 135 150 165 180 195 210 225 240 255 270 285 300 Days

L1 L2 L3 L1R L2R L3R

144

Fig. 7. Dynamic of the growth of H. elegans per instar in a Ultisol soil, with and without wheat roots .

900 ) )

800 mg

mg 700

( ( 600 500

weight weight 400

300 Mean Mean Mean 200 100

30 45 60 75 90

105 120 135 150 165 180 195 210 225 240 255 270 285 300 Days

L1 L2 L3 L1R L2R L3R

145

Fig. 8. Larval stage development of H. elegans in Inceptisol type soil, with and without wheat roots. A: without roots; B: with roots.

100

A 90 80

60 50 40 30

20 (%) (%) 10

15 30 45 60 75 90

120 135 150 165 180 195 210 225 240 255 270 285 300 315 105

L1 L2 L3 P A porcentage porcentage 100

90 Instar Instar Instar 80

70 60 50 40

B 10

15 30 45 60 75 90

120 135 150 165 180 195 210 225 240 255 270 285 300 315 105 Days

146

Fig. 9. Larval stage development of H. elegans in Andisol type soil, with and without wheat roots. A: without roots; B: with roots.

100

A 90

60 50 40 30

20 (%) (%) 10

15 30 45 60 75 90

120 135 150 165 180 195 210 225 240 255 270 285 300 315 105

L1 L2 L3 P A

porcentage porcentage 100

90 Instar Instar Instar 80 70 60 50

20 B 10

15 30 45 60 75 90

120 135 150 165 180 195 210 225 240 255 270 285 300 315 105 Days

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Fig. 10. Larval stage development of H. elegans in Ultisol type soil, with and without wheat roots. A: without roots; B: with roots.

100

A 90

70 60 50 40 30

20 (%) (%) 10

15 30 45 60 75 90

120 135 150 165 180 195 210 225 240 255 270 285 300 315 105

L1 L2 L3 P A porcentage porcentage 100

90 Instar Instar Instar 80 70

50 40 30

10 B 0

15 30 45 60 75 90

105 120 135 150 165 180 195 210 225 240 255 270 285 300 315

Days

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9. EFECTO DEL TIPO DE SUELO Y LAS RAICES SOBRE EL CRECIMIENTO Y DESARROLLO DE LAS LARVAS. Phytholaema herrmanni Germain (SCARABAEIDAE: MELOLONTHINAE).

Este artículo puede ser consultado bajo el título:

Cisternas, A. E.; Carrillo, Ll. R. y Millas, P. FITNESS OF Phytholaema herrmanni Germain (COLEOPTERA: SCARABAEIDAE: MELOLONTHINAE) LARVAE, REARED IN DIFFERENT SOIL TYPES WITH AND WITHOUT ROOTS. Chilean Journal of Agricultural Research. Code: CJAR 11014 (Submitted)

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FITNESS OF Phytholaema herrmanni Germain (COLEOPTERA: SCARABAEIDAE: MELOLONTHINAE) LARVAE, REARED IN DIFFERENT SOIL TYPES WITH AND WITHOUT ROOTS.

Ernesto Cisternas1*; Roberto Carrillo2 and Paz Millas1

1 Graduate School, Faculty of Agricultural Sciences, Universidad Austral de Chile. 2 Laboratory of Entomology, Institute of Production and Plant Protection, Universidad Austral de Chile, P.O. BOX 567 Valdivia, Chile. * [email protected]

ABSTRACT

The growth and development of Phytholaema herrmanni Germain (Coleoptera: Scarabaeidae: Melolonthinae) larvae were studied in three soil types, with and without the presence of roots. Larval survival, growth and development were affected by soil types. The survival and growth of P. herrmanni were higher in Inceptisol soil type than in Andisol and Ultisol soil types. The L1 and L2 instars of larva reared in the different soil types tested with roots were shorter compared to those of larva maintained in soils without roots. Results confirm the higher nutritional requirements of Melolonthinae compared to Rutelinae. Despite the larval weights reached in soils with roots and in Inceptisol soil without roots, no larvae were able to pupate under these conditions.

Key words: Scarabaeidae, Melolonthinae, P. herrmanni , larval growth, survival.

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INTRODUCTION

Phytholaema herrmanni Germain is one of the most common species in the scarabaeid larval assemblage that consumes roots of the different plant species that make up pasture lands in southern Chile, as well as other arbustive and arboreal species of economic importance (Durán, 1952; Durán, 1954; Carrillo, 1986; Artigas, 1994; Aguilera et al., 1996; Klein and Waterhouse, 2000). The effects of herbivory on roots and soil type on the growth and survival of P. herrmanni larvae has been unknown. The species is endemic, with a distribution in Chile between the Bio Bio and Los Lagos Regions (Prado, 1991; Artigas, 1994; Klein and Waterhouse, 2000). It has an annual cycle with periods of seasonal flights that vary depending on the region and climatic conditions. In the Araucania Region, the adult flight is between the 34th and 40th week (Durán, 1954).

Abiotic factors can affect the survival, growth and development of larvae in the soil (Kard et al., 1988; Villani and Wright, 1988), as well as biotic factors, such as diseases caused by bacteria, fungus, protozoa, viruses, nematodes and other agents (Glare and Jackson, 1992). Feeding during the larval stage can have important effects on the development of scarabaeid populations, in particular on those species that do not feed as adults (Logan et al., 2001), as in the case of P. herrmanni, Durán, (1954).

The quality of food consumed during the larval period also influences morpohology, growth, reproduction and survival of the insects (Logan et al., 2001; Logan and Kettle, 2002; Karino et al., 2004). Larvae bred under a regime of poor nutrition reduce the duration of the larval stage and pupate prematurely, leading to the early emergence of small adults (Moczek, 1998; Shafiei et al., 2001). In other cases, the duration of the larval stage is extended (Esperk, et al., 2007). The potential fecundity of species that only feed at the larval stage is related to larval feeding (Logan et al., 2001).

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The presence or absence of roots is an important factor for the adequate development of some species (Farell, 1972; Ridsdill Smith, 1975; Berry and Potter, 1995; Logan and Kettle, 2002). The root exudates of the plants do not produce variations in the growth and survival of third instar larvae of P. herrmanni (Rojas, 2005). The majority of insects that feed on roots are considered polyphagous herbivores. Nevertheless, some exhibit preferences for certain plant species, confirming the existence of favorable and unfavorable species (East and King, 1977; King et al., 1981; Prestidge et al., 1985). Physical and chemical characteristics of the soil, such as OM, pH, moisture, temperature, structure, porosity and aeration also affect invertebrates (Radcliffe, 1971; Ridsdill Smith et al., 1975; Vitum and Tashiro, 1980; Régnière et al., 1981; Potter and Gordon, 1984; Vitum, 1984; Curry, 1987; Brown and Gange, 1990). Artificial fertilizers had little or no effect on the growth and survival of Costelytra zealandica (White) (Prestidge et al., 1985).

Ritcher, (1958) adopted the classification of scarabaeid larvae according to their feeding habits proposed by De Fluiter, indicating that larvae that feed only on dead organic matter are Cetoniinae. Larvae that consume organic matter, but in its absence consume roots, belong to the sub-families Rutelinae and Dynastinae, and larvae that mainly consume plant roots belong to the Melolonthinae.

The objectives of this study were to establish the effect of the three most important soil types in southern Chile, with and without roots, on the biological potential of the species, measured through the survival, development and growth of the larval stage of P. herrmanni and the formation of pupae and adults.

MATERIALS AND METHODS

Insects. Adult P. herrmanni were collected in the field at the Universidad Austral de Chile’s Santa Rosa Experimental Station (39°47’S and 73°14’ W). The adult insects were conserved for 10 days in plastic containers (10 L), with Andisol soil type of the Valdivia

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series. The eggs obtained every 48 hours were transferred to 1 L terrariums with soil, maintained at a constant temperature of 18 ± 2 ºC. Beginning on the day 15 of incubation, the terrariums were examined every 24 h to obtain neonate larvae (L1). Each L1 larvae obtained in the laboratory was carefully handled and selected for its physical condition and its ability to enter the soil. Once larval weight was registered with an analytic scale (Precisa 100A-300M) the selected larvae were placed individually in 100 mL polypropylene pots with ventilated covers. At 24 hours, the L1 larvae that had not dug into the soil on their own, were replaced. The treatments were kept under constant laboratory breeding conditions, with a temperature of 18 ± 2 ºC, a relative humidity of 50±10% and a photoperiod of (0L:24D). Every 15 days, the live larvae were weighed and the larval instar and mortality were registered for up to 420 days. The relative growth rate was also determined (TCR) = (LN Final weight (mg) – LN Initial Weight (mg)) /number of days between observations (Adams and Van Emden, 1972).

The plants of Triticum aestivum cv. Kumpa, in densities of 4 plants per pot, provided living roots. The plants were seeded four days before the beginning of the experiment and before each biweekly evaluation. Soil compaction in the pots ranged between 1.25 and 1.50 kg cmˉ2, determined with a manual penetrometer (Pocket penetrometer CL-700 / SOILTEST INC., Chicago, USA).

Soils Soils were collected in different localities in the Los Lagos and Los Ríos Regions. 1. Inceptisol (Huiño Huiño, soil series), Aquandic Humaquept: (40°43’S and 72°48’W) 139 msnm; 2. Ultisol (Huilma, soil series), Andic Palehumults: (40°47’S and 73°16’W) 108 msnm; 3. Andisol (Puerto Fonk, soil series), Pachic Melanudands: (40°39’S and 72°39’W) 219 msnm Ciren, (2003). The soils were maintained with a humidity level of 35 ± 5 % p/p. When the weight loss in the pots exceeded 10 %, the weight was restored with distilled water.

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Chemical analysis of the soil, POM and basal respiration

Each soil was chemically analyzed at the Soil Laboratory of the Universidad Austral de Chile. The soils used were characterized by high organic matter content, low pH and available phosphorous, together with a high K content and aluminum saturation (Table 1).

The organic matter in each of the soils was divided according to the size of particulate organic matter (POM) and analyzed to establish the percentage of matter larger than 53 µm. To do this, some 10 g were sieved at 2 mm, dried for 48 h at 30ºC and dispersed in a 30-L solution of sodium hexametaphosphate (5 g Lˉ¹) agitated for 15 hours in reciprocal agitator at 140 rpm. The dispersed samples were separated by a 53 µm sieve with several rinsings, until the distilled water ran clear. The material retained in the sieve and the fraction that passed through the sieve were dried at 45ºC for 48 h and subsequently C and N were determined (Cambardella & Elliot, 1992) (Table 2).

The basal respiration of the soil was determined by measuring released CO2 using the Isermyer method. To do this, 10 g samples of dried fine soil were humidified at 50% gravimetric humidity. The soil was enveloped in a gas that was suspended with threads within a 1 L glass container with 25mL of baryta water (7.17 g Ba (OH2) · 8 H2O + 1 g

BaCl2, dissolved in a liter of distilled water. The glass flask was rapidly and hermitically sealed. Parallel to this, a control was established without soil. After 24 h of incubation at

28ºC, the barium hydroxide that did not react with CO2, was tittered with HCL (0.1 N) adding some drops of phenolphthalein (Steubing et al., 2002). The soils used in the research presented high levels of basal respiration, which concurs with the values for volcanic soils, as indicated by Zagal, et al. (2002), noting the Inseptisol that presented a markedly higher level of basal respiration, in the measurement taken at 24 hours, which was at least 33% higher than the levels of the other two soils (Table 3).

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Data analysis

The experimental design was a completely random factorial arrangement (3x2), three soil types and two conditions, with and without roots. Each treatment had 60 replicates. An ANOVA was used (PROC GML, SAS Institute, 2009) for repeated measurements where the insect nested with the soil/treatment was considered as an experimental error to partially remove the correlation upon repeatedly measuring the weight of the insect over time.

RESULTS

Survival of the larvae of Phytholaema herrmanni

The survival of the larval stage of P. herrmanni is presented in (Fig. 1). Under the conditions of the study, P. herrmanni, in Iceptisol soil without roots was able to grow and develop for a period of 420 days, while in the same soil with roots; they grow over a period of 405 days. In the Andisol without roots, larvae only survived 30 days and in the soil with roots, for 345 days. In the Ultisol without roots, the larvae survived 345 days, and with roots for 375 days. At 105 days, 25 % of the evaluation period, the highest survival of P herrmanni was 68.3 % in the Inceptisol soil without roots, and in a lower percentage, 56.7 % in the same soil with roots. In the Andisol without roots the survival rate was 0% and in the same soil with roots was 5.0 %. In the Ultisol soil without roots the survival rate was 35.0% and in the same soil with roots it was 51.7%. At 210 days, 50% of the evaluation period, the survival rate in the Andisol with roots was 3.3 %. The larvae bred in Inceptisol and Ultisol soils without roots presented survival rates of 41.7 % and 16.7%, respectively. The survival rates in Inceptisol and Ultisol soils with roots were 50.0% and 35.0 % respectively. At 315 days, 75% of the evaluation period, the highest survival rate of P. herrmanni was 30.0 % and 36.7% in the Inceptisol soil with and without roots. The survival

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rates in the Ultisol soil with and without roots were 26.7% y 10.0% respectively and in the Andisol soil with roots it was only 3.3%.

Growth of the larvae of P. herrmanni

Relative growth rates

The relative growth rates (RGR) of the P. herrmanni larvae according to the soil type can be seen in (Fig. 2, 3 and 4). The larvae bred in the Inceptisol soil had similar RGR, with and without roots, even though in the Andisol the larvae without roots did not survive and those with roots showed a similar RGR to that of larvae bred in the Inceptisol with roots. The RGR of larvae in Ultisol soil without roots, between days 75 and 90, and from days 180 to 390 presented differences from the larvae bred in the same soil with roots (P<0.05). The larvae in the three types of soil bred with roots presented similar RGR.

Larval growth by instar

The effect of different soils with and without roots on larval growth was estimated through biweekly weight per instar. The larvae of the first instar bred in the three soils were all different from each other (P<0.05) and growth in the presence of roots was different from that of larvae bred in soils with roots (P<0.05). The soil-roots interaction was significant (P<0,05). The larvae of the second instar did not show differences between the Inceptisol and Ultisol soil, but did with the Andisol, as well as significant differences with and without roots (P<0.05). The soil-roots interaction was significant (P<0.05), Table 4. The larvae of the third instar showed differences between the Inceptisol soil and the Ultisol and Andisol soils, and significant differences between with and without roots (P<0.05).

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Larval growth dynamic

The larvae only reached the L3 instar in the Inceptisol and Ultisol soils with and without roots, while larvae in Andisol soil only reached the same instar in soil with roots. Nevertheless, the L3 instar did not reach to the pupal stage in any of the soils, with and without roots. The larvae bred with roots in Inceptisol soil (Fig.5) maintained continued growth for 285 days, while those bred in Ultisol and Andisol soils (Fig. 6 and 7) continued for 255 days. However, in the larvae bred without roots in Inceptisol and Ultisol soils, growth lasted 270 and 300 days, respectively. In the Andisol soil without roots, the larvae did not develop. Up to 405 days, the larvae bred in Inceptisol, with and without roots, continuously reduced their average weight from 409.65 mg to 270.15 mg and from 326.85mg to 253.2 mg, respectively; but in the condition without roots, by 420 days larval had decreased dramatically to 94.9 mg. The maximum weight of continuous growth reached in the Ultisol soil without roots at 300 days (192.8 mg) and then decreased to 179.2 mg by 345 days The larvae bred in Andisol with roots reached the average maximum growth at 255 days, with a weight of 269.9 mg, which began to decrease rapidly at 345 days to 101.6 mg. The average maximum weight reached by the L3 larvae with and without roots was higher in Inceptisol than in Ultisol and Andisol soils. The average weights determined in L3 larvae were different in Inceptisol with roots (328.57 mg) and without roots (254.49 mg). In the Ultisol without roots, the average L3 weight was 254.81 mg and with roots it was 134.88 mg. The average larval weight in the Andisol with roots, 201.43 mg, was lower than that of larvae in other soils (Table 5).

Development of the larvae

The larvae of P. herrmanni did not complete their larval development and transform into pupae, whether in Inceptisol, Andisol or Ultisol soils, with and without roots (Fig. 8, 9 and 10). In the three soil types with roots and in Inceptisol and Ultisol soils without roots, larval development reached the L3 instar. In the Inceptisol soil without roots, the L1 instar

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developed in a period of between 45 and 75 days and the L2 instar between 45 and 225 days. In this same soil with roots, the L1 instar developed in a period between 45 and 60 days and the L2 instar between 45 and 150 days. In the Andisol soil without roots, the L1 instar did not complete its development, while in the same soil with roots, the L1 instar developed between 45 and 60 days and the L2 instar developed between 45 and 150 days. The lapse for the developments of the L1 and L2 instars was similar in the three instars with roots. In the soils without roots, development was slower and the lapses were longer than with roots. The development rates of larvae according to soil type and the presence of absence of roots were different (P<0.05).

DISCUSSION

The results obtained confirm what was indicated by Ritcher, (1958) regarding the need of Melolonthinae to feed on plants, given that the growth, development and survival of the larvae were different with and without the presence of plants. The differences were more marked than in a Rutelinae, which was kept under similar conditions. This reaffirms the distinct nutritional requirements of the larvae of these sub-families and the probable higher nutritional requirements of Melolonthinae. Moreover, the results obtained for P. herrmanni concur in part with the results obtained in Australia for another Melolonthinae, Sericesthis nigrolineata Boisd., determining that the relative growth rate was higher in the treatments with live roots (Ridsdill Smith, 1975). The larvae de P. herrmanni did not survive in Andisol soil in the absence of roots. In the Inceptisol and Ultisol soils with and without roots and the Andisol soil with roots, the larvae could survive, grow and develop, but did not succeed in passing from the L3 instar to the pupal stage in any of the soils, with or without roots. The fact that the larvae did not reach the pupal stage, which has occurred in similar assays in which plants were maintained throughout the period (Pezoa, 1996), indicates two possibilities first of all that the roots, which were renewed every 15 days, did not constitute an important part of the diet in relation to the soil, given the limited development. Consequently, although root matter represented an important nutritional

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contribution, it was not sufficient, and the larvae would need to consume large quantities of radical matter (Bauchop and Clarke, 1977) or that the nutritional composition of new roots is not adequate to meet the needs of the larvae of this species. In other assays, Rojas, (2005) used sheep manure in a 1:1 mixture with soil without roots, obtaining a higher survival rates and higher weights of L2 and L3 larvae in sheep manure with soil without roots, obtaining the highest survival rate and the highest weight of the L2 and L3 larvae at 107 days 58 % and 311 mg on comparing with based soil 0% and 42 mg and with soil with different types of roots an average of 25.8% and 147 mg. Rojas, (2005) established that the roots of different species of plants are not capable sources for providing the necessary nutrients to allow for adequate growth, in contrast to what occurs when the manure is added to the soil, probably owing to the high microbial load in the manure, which constitutes an important part of the supply of nutrition and energy for the larvae of this species.

Larval feeding can have important implications in the development of scarabaeid populations, in particular when the species does not feed as an adult. Through this study, it has been shown that although P. herrmanni responds to the soil type and the presence of roots, these are not sufficient to supply the nutritional requirements to complete its growth and development until the adult stage. Rojas, (2005) established that there are no significant differences in weight between larvae of P. herrmanni bred with legume roots and those bred with grasses roots. According to Saeki et al. (2005), the size of the female influences the biotic potential and male mate preference in P. japonica. Shafiei et al.(2001) established that in the dung beetle Onthophagus taurus, the larvae in a poor nutritional state have shorter larval periods and produce smaller adults. In contrast, the larval period of female larvae of Allomyrina dichotoma L. in poor nutritional condition is extended (Karino et al., 2004). Larval feeding and nutrition influences larval survival and growth, as well as the size of adults, which in turn affects the biotic potential, which can be an important factor in population dynamics (Moczek, 1998; Logan et al., 2001; Shafiei et al., 2001; Logan and Kettle, 2002; Karino et al., 2004; Chown and Gaston, 2010). In this study, the larvae of P. herrmanni reduced their weight and increased the length of the larval stage, not

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reaching molting to complete their development as larvae. The growth rate of each molting is determined by the size and number of cells and by well known hormonal events (Chown and Gaston, 2010). An inadequate hormonal balance could explain this behavior in the larvae, mainly because of nutritional deficiences (Nation, 2002; Mirth and Riddiford, 2002).

The Inceptisol soil type presented better conditions for the growth, development and survival of the larvae of P. herrmanni independent of the presence or absence of roots. The higher quality of this soil favors the biological potential of the larvae and is probably associated with the higher microbiological activity found in this type of soil, measured with basal respiration. This study found 33% more basal respiratory activity in Inceptisol soil (Table 3). The higher basal respiration is due to the higher level of activity of microorganisms. Soil factors like aluminum saturation and exchangeable aluminum are considered adequate in Inceptisol soil, while in Andisol and Ultisol soils they are considered detrimental to plant growth and affect the type and quantity of microorganisms in the soil (Kanazawa et al., 2005). This suggests that labile organic matter and microorganisms did not provide all the nutritional requirements of P. herrmanni larvae, while larvae of other insects do not complete their development in the absence of certain amino acids, lipids, sterols, polyunsaturated fatty acids or vitamins (Nation, 2002). The P. herrmanni larvae were affected by the soil type and by the absence of roots. They survived better and gained more weight when they feed on live roots, compared to developing in soils without roots. This is similar to what was indicated by Logan and Kettle, (2002) for the species Dermolepida albohirtum (Waterhouse) and by Ridsdill Smith, (1975) for Anisoplia austriaca (Herbst.), Popillia japonica Newmann, Costelytra zealandica White and Sericesthis geminata (Boisduval), the exception being Rhopacea morbillosa Blackburm.

P. herrmanni is capable of feeding on organic matter present in the soil, but it evident that it needs the presence of roots and other OM substrates to complete its development. If the

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soil is adequate and there are no roots, P. herrmanni larvae can grow to a lesser extent and extend its larval period in Inceptisol and Ultisol soils. In contrast, in Andisol soil it develops to L1. According to Ridsdill, (1975), many scarabaeid species can feed on OM and live roots, but their selective abilities are not well understood. The response thus to the ability to select roots as a food source would be intrinsic, depending on factors, proof of what was indicated by Berry and Potter (1995) who determined the effect of weeds on the survival and growth of P. japonica and Cyclocephala lurida Bland suggesting that the latter is not so dependent on live roots as P. japonica.

The greater biological potential that larvae present in Inceptisol soil is in contrast to the small populations that this type of soil presents, probably owing to the catastrophic effects produced by periodic flooding that occur during the winter season in this type of soil and because of the depth of these soils, considering that this species constructs vertical galleries that are over 25 cm in depth. These catastrophic effects have been reported in other insect groups, resulting in important reductions in the density or even the extinction (Hanski, 1998; Leibold, et al., 2004).

According to Ritcher, (1958), Melolonthinae larvae feed on humus and living plant tissue. Through this study it has been shown that the OM present in the soil and living plant tissue, such as roots, were not sufficient for the development of the Melolonthinae P. herrmanni. In the case of this species, it is necessary to ask from where it obtains the nutrients to complete its development. The answer could be in its movement in the soil profile as part of its feeding behavior. This species is capable of reaching the soil surface when constructing its vertical gallery under the soil, raising the soil and covering the necks of plants. Because of this, it is highly probable that they feed on surface residues. At the bottom of the gallery it is possible to find small vegetal remains that are probable not there was accident (Cisternas, unpublished).

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RESUMEN

Adecuación biológica de las larvas de Phytholaema herrmanni Germain (Coleoptera: Scarabaeidae: Melolonthinae), criadas en diferentes tipos de suelo con y sin raíces. El crecimiento y desarrollo de las larvas de Phytholaema herrmanni Germain (Coleoptera: Scarabaeidae: Melolonthinae) fue estudiado en tres tipos de suelo, con y sin la presencia de raíces. La sobrevivencia, el crecimiento y el desarrollo larval fue afectada por los tipos de suelo. La sobrevivencia y crecimiento de P. herrmanni fue mayor en el suelo tipo Inceptisol que en los suelos tipo Andisol y Ultisol. La duración del estadio L1 y L2 de las larvas criadas en los diferentes tipos de suelos evaluados con raíces fueron más cortas comparados con aquellas larvas mantenidas en suelos sin raíces. Los resultados confirman el mayor requerimiento nutricional de los Melolonthinae comparado a los Rutelinae. A pesar del peso larval alcanzado en los suelos con raíces y en el suelo tipo Inceptisol sin raíces, las larvas no fueron capaces de pupar bajo esas condiciones.

Palabras clave: gusano blanco, Phytholaema herrmanni, crecimiento larval, desarrollo larval, suelo-raíz, sobrevivencia

CONCLUSIONS

The P. herrmanni larvae were affected by the soil type and by the absence of roots, they survived better and gained more weight when they feed on live roots, compared to developing in soils without roots.

The larvae de P. herrmanni did not survive in Andisol soil in the absence of roots but in the Inceptisol and Ultisol soils with and without roots and the Andisol soil with roots, the larvae could survive, grow and develop, but did not succeed in passing from the L3 instar to the pupal stage in any of the soils, with or without roots.

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Through this study, it has been shown that although P. herrmanni responds to the soil type and the presence of roots, these are not sufficient to supply the nutritional requirements to complete its growth and development until the adult stage.

The larvae of P. herrmanni reduced their weight and increased the length of the larval stage, not reaching molting to complete their development as larvae. P. herrmanni is capable of feeding on organic matter present in the soil, but it evident that it needs the presence of roots and other OM substrates to complete its development. If the soil is adequate and there are no roots, P. herrmanni larvae can grow to a lesser extent and extend its larval period in Inceptisol and Ultisol soils. In contrast, in Andisol soil it develops to L1.

Through this study it has been shown that the OM present in the soil and living plant tissue, such as roots, were not sufficient for the development of the Melolonthinae P. herrmanni.

ACKNOWLEDGEMENTS

We are grateful to Sonia Santana H., Jorge Barría M. and Manuel Muñoz, owners of the farms where soil samples were obtained. We wish to thank the personnel of the Soil Laboratory at the Universidad Austral de Chile. We also thank Gustavo Valdebenito for his assistance in the field, Leticia Silvestre for her assistance in the laboratory, and Hector Uribe for advice on statistics.

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Table 1. Chemical characteristics of the soils used

Parameters Inceptisol Andisol Ultisol

pH in water (1:2.5) 5.7 5.5 5.6

pH CaCl2 (1:2.5) 5.1 4.7 4.8 Organic matter (%) 15.2 22.2 16.6 (ppm N-

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Table 2. Particulate matter content of organic matter (POM) greater than 53 µm in the studied soils.

Soil type POM > 53 µm (%) Recovery (%) Inceptisol 5.82 ± 0.06 b 97.36 Andisol 6.56 ± 0.06 a 98.03 Ultisol 6.56 ± 0.19 a 94.80

Data from the same column with different letters are significantly different at P< 0.05 as tested with LSD Multiple Range test.

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Table 3. Basal respiration of the soils (CO2 mg. h-1.kg dry soil-1)

Parameter Hours Inceptisol Andisol Ultisol

24 0.36 a 0.24 b 0.23 b Respiration 48 0.26 a 0.19 b 0.13 b

Data from the same row with different letters are significantly different at P< 0.05 as tested with LSD Multiple Range test.

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Table 4. Factorial analysis of variance (ANOVA) with repeated measurements for larval live weight per instar of P. herrmanni .

Instar Source Df SS F P Soil 2 12149.45 160.08 < 0.0001 I Roots 1 3893.12 102.59 < 0.0001 Soil x roots 2 2099.14 27.66 < 0.0001 Soil 2 20539.31 8.61 0.0003 II Roots 1 20554.68 17.23 < 0.0001 Soil x roots 2 24175.68 20.27 < 0.0001 Soil 2 1166421.79 19.34 < 0.0001 III Roots 1 1930768.16 64.02 < 0.0001 Soil x roots 2 190716.01 6.32 0.0137

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Table 5. Average weight per larval stage of P. herrmanni in three soil types, with and without roots.

Soil Inceptisol Andisol Ultisol Roots Without With Without With Without With Instar Mean ± SD Mean ± SD Mean ± SD L1 17.45 ± 9.53 19.54 ± 11.95 6.45 ± 1.79 10.45 ± 6.48 14.28 ± 6.49 19.87 ± 12.84 L2 72.11 ± 26.14 73.12 ± 27.36 48.18 ± 21.76 43.97 ± 5.78 77.89 ± 23.71 L3 254.49 ± 83.94 328.57 ± 100.46 201.43 ± 73.31 254.81 ± 76.91

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Figure 1. Survival of P. herrmanni larvae bred in three soils, with and without wheat roots (I: Inceptisol; IR: Inceptisol roots; A: Andisol; AR: Andisol roots; U: Unltisol; UR; Ultisol roots).

100 90 80 70 60 50

40 Survival (%) 30 20 10

30 60 90

120 150 180 210 240 270 300 330 360 390 420 Days

I IR A AR U UR

174

Figure 2. Relative growth rates (RGR) of P. herrmanni larvae in Inceptisol type soil, with and without wheat roots.

7,0

6,0

day day

/ / /

5,0

mg mg / / /

4,0 mg mg

3,0 larvae

larvae

of of

. . .

larvae of mg/mg/day R.G.R. 2,0 R.G.R R.G.R 1,0

0,0

15 30 45 60 75 90

120 135 150 165 180 195 210 225 240 255 270 285 300 315 330 345 360 375 390 405 420 105 Days

No roots Roots

175

Figure 3. Relative growth rates (RGR) of P. herrmanni larvae in Andisol type soil, with and without wheat roots.

7,0

6,0

day day / / /

5,0

mg mg / /

/ mg mg 4,0

larvae

larvae 3,0

of of . . . R.G.R. of larvae of mg/mg/day R.G.R. 2,0

R.G.RR.G.R 1,0

0,0

15 30 45 60 75 90

105 120 135 150 165 180 195 210 225 240 255 270 285 300 315 330 345 360 375 390 405 420 Days No roots Roots

176

Figure 4. Relative growth rates (RGR) of P. herrmanni larvae in Ultisol type soil, with and without wheat roots.

7,0

6,0

day day / / /

5,0

mg mg / /

/ mg

mg 4,0 larvae

larvae 3,0

of of . . .

R.G.R. of larvae of mg/mg/day R.G.R. 2,0 R.G.R R.G.R 1,0

0,0

15 30 45 60 75 90

105 120 135 150 165 180 195 210 225 240 255 270 285 300 315 330 345 360 375 390 405 420 Days No roots Roots

177

Figure 5. Dynamic of the growth of P. herrmanni per instar in a Inceptisol soil, with and without wheat roots.

600

500 )

)

mg mg ( ( 400

weight weight 300

larvae larvae 200

weight (mg) larvae Average Average Average Average 100

15 30 45 60 75 90

105 120 135 150 165 180 195 210 225 240 255 270 285 300 315 330 345 360 375 390 405 420 Days

L1 L2 L3 L1+R L2+R L3+R

178

Figure 6. Dynamic of the growth of P. herrmanni per instar in an Andisol soil, with and without wheat roots.

600

500 )

)

mg mg ( ( 400

weight weight 300

larvae larvae

200 Average larvae weight (mg) larvae Average

Average Average Average 100

30 45 60 75 90

105 120 135 150 165 180 195 210 225 240 255 270 285 300 315 330 345 360 375 390 405 420 Days L1 L2 L3 L1+R L2+R L3+R

179

Figure 7. Dynamic of the growth of P. herrmanni per instar in a Ultisol soil, with and without wheat roots .

600

500 )

)

mg mg ( ( 400

weight weight 300

larvae larvae

200 Average larvae weight (mg) larvae Average

Average Average Average 100

30 45 60 75 90

105 120 135 150 165 180 195 210 225 240 255 270 285 300 315 330 345 360 375 390 405 420 Dias L1 L2 L3 L1+R L2+R L3+R

180

Figure 8. Larval stage development of P. herrmanni in inceptisol type soil, with and without wheat roots. A: without roots; B: with roots.

100 A 80

(%) (%) 20

15 30 45 60 75 90

120 135 150 165 180 195 210 225 240 255 270 285 300 315 330 345 360 375 390 405 420 B 105

100 porcentage porcentage

80 Instar Instar Instar Instar

40 20 B 0

15 30 45 60 75 90

105 120 135 150 165 180 195 210 225 240 255 270 285 300 315 330 345 360 375 390 405 420

Days L1 L2 L3

181

Figure 9. Larval stage development of P. herrmanni in Andisol type soil, with and without wheat roots. A: without roots; B: with roots

100 A 80

40 (%) (%)

15 30 45 60 75 90

105 120 135 150 165 180 195 210 225 240 255 270 285 300 315 330 345 360 375 390 405 420

porcentage porcentage

100 Instar Instar Instar 80

20 B

15 30 45 60 75 90

120 135 150 165 180 195 210 225 240 255 270 285 300 315 330 345 360 375 390 405 420 105 Days L1 L2 L3

182

Figure 10. Larval stage development of P. herrmanni in Ultisol type soil, with and without wheat roots. A: without roots; B: with roots.

100 A 80

40 (%) (%) 20

15 30 45 60 75 90

120 135 150 165 180 195 210 225 240 255 270 285 300 315 330 345 360 375 390 405 420

105 porcentage porcentage 100

80 Instar Instar Instar Instar 60

20 B

15 30 45 60 75 90

120 135 150 165 180 195 210 225 240 255 270 285 300 315 330 345 360 375 390 405 420 105 Days L1 L2 L3

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10. DISCUSIÓN GENERAL

Un gran número de especies de escarabajos, tales como Hylamorpha elegans (Burm.) (Scarabaeidae: Rutelinae), Phytholoema herrmanni Germain, (Scarabaeidae: Melolonthinae) y otros como Sericoides germaini Dalla Torre, Tamarus villosus Burm., Athlia plebeja Burm., Athlia rustica Erichson, Brachysternus prasinus Guérin-Méneville, Pristerophora picipennnis (Solier), Schizochelus modestus Philippi, Sericoides viridis Solier , Sericoides obesa Germain, Aulacopalpus punctatus (Fairmaire and Germain), son en mayor o menor magnitud plagas en Chile (Prado, 1991; Klein y Waterhouse, 2000). Los adultos de casi la totalidad de especies se alimentan del follaje de algunas plantas; las que por lo general, presentan daños menores. Sin embargo, estos perjuicios en algunas temporadas son significativos en especies forestales como Nothophagus obliqua (Mirb.) Oerst . Las larvas de estas especies se alimentan bajo el suelo de los materiales que lo constituyen como MO y estructuras de las plantas, siendo las raíces las más utilizadas como alimento. La mayoría de estas especies producen daños significativos en praderas, cultivos anuales y plantaciones de frutales menores y especies forestales nativas e introducidas (Durán, 1952; Durán, 1954; Durán, 1976; Prado, 1991; Klein y Waterhouse, 2000).

Las características morfológicas de los adultos permiten su identificación y conocer su distribución y abundancia a través del país. Sin embargo, como en muchos grupos de insectos, se desconoce las características morfológicas de los estados inmaduros de las distintas especies, esto hace difícil realizar estudios en profundidad de los ensambles de larvas de escarabaeidos. Es por ello, que este estudio es un primer paso del autor, para poder diferenciar las especies que componen estos ensambles. Las descripciones taxonómicas realizadas de las larvas de siete especies P. picipennis, Sch. modestus, P. herrmanni, P. dilutipes, A. mahdii, H. elegans, A. punctatus representan un avance importante en el conocimiento. Estos antecedentes son esenciales para desarrollar los estudios biológicos, necesarios para comprender y establecer la importancia de cada

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una de las especies en los ensambles que ellas conforman. Asi mismo, permitir el desarrollo de estrategias de manejo adecuadas a los diferentes cultivos. En la actualidad, con la información generada en esta investigación y los estudios que el autor continua desarrollando, sobre la taxonomía de las larvas de las especies en Chile, será posible en un período corto de tiempo diferenciar morfológicamente a un número importante de las principales especies de escarabaeidos en su estado larval, presentes en la zona centro sur y sur del país.

El género Phytholaema Blanchard se encuentra únicamente en la región Netropical e incluye a tres especies Phytholaema dilutipes (Fairmaire & Germain ), Phytholaema herrmanni Germain, con dos subespecies P. herrmanni herrmanni Germain y P. herrmanni pallida Saylor y Phytholaema mutabilis (Solier) (Evans y Smith 2009). Todas ellas son encontradas en Chile, sólo P. mutabilis es también hallada en Argentina. De acuerdo a Evans y Smith (2009) la posición tribal de Phytholaema es incertae sedis, es decir se necesita más información para ubicar tribalmente este género.

Diferencias morfológicas bien definidas entre los terceros estadios de P. herrmanni y P. dilutipes son presentadas en esta tesis. Ambas especies tienen una abertura anal en forma de Y, con dos pallidia paralelas y longitudinales. P. herrmanni tiene 9-16 pali, mientras que P. dilutipes tiene 11-19 pali. El tegillum de P. herrmanni tiene 12-22 setas curvas y P. dilutipes tiene 19-28 setas curvas. El septulum es más amplio en P. herrmanni, pero más angosto en P. dilutipes. Los espiráculos en A6, A7 y T son similares en tamaño y A8 conspicuamente más pequeño en P. herrmanni; los espiráculos A6 y A8 son conspicuamente más pequeños que A1-A5 y hasta A7 y T en P. dilutipes. El plato respiratorio en el espiráculo toráxico de P. herrmanni tiene 9-12 largas aberturas irregulares a través de su diámetro, mientras en P. dilutipes, el plato respiratorio tiene 12-15 pequeñas aberturas irregulares a través de su diámetro. El área estriduladora maxilar de P. herrmanni esta formada por una fila de 10-14 dientes redondeados y en P. dilutipes el área estriduladota esta formada por una fila de 9-11 dientes redondeados.

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El género Arctodium Burmeister, 1844 se distribuye en la región Neotropical y esta representado por cuatro especies: A. discolor Erichson, A. mahdii Hawkins, A. planum Blanchard and A. vulpinum Erichson (Hawkins, 2006; Evans y Smith, 2009). Estas especies se encuentran sólo en Chile desde la Región de Coquimbo a la Región de La Araucanía. La morfología de las larvas de la tribu Lichniini es desconocida dado a que ninguna de las larvas ha sido descrita (Hawkins, 2006). La colecta de la especie en localidades de la Región de Los Ríos amplía el área de distribución dada por Hawkins, (2006). Su importancia en los ensambles de gusanos blancos es desconocida, situación que cambiará como resultado de esta investigación.

La larva de A. mahdii tiene una abertura anal en forma de Y, sin palidium. El tegillum esta cubierto por largas setas delgadas. Los espiráculos abdominales A1-A3 son de igual tamaño y A5–A8 son de igual tamaño entre sí; pero conspicuamente más pequeños que los espiráculos anteriores y las concavidades de sus platos respiratorios esta dirigida ventralmente. El antenito apical tiene una mancha sensorial dorsal y dos manchas sensoriales ventrales. La chaetoparia tiene dos filas definidas de setas gruesas formando un arco. Las mandíbulas tienen un acia bien desarrollada, siendo más largas que anchas y sin área estriduladora. El palpo maxilar es muy curvado desde una vista lateral. La galea y lacinia están separadas. Cada tarsungulus tiene dos setas proximales.

Hylamorpha elegans (Burmeister, 1844) y Aulacopalpus punctatus (Fairmaire & Germain, 1860) son miembros de géneros endémicos de Sudamérica. H. elegans es la especie más común en varios ecosistemas y tiene una amplia distribución en Chile (Durán, 1952; Carrillo y Cerda, 1987; Ratcliffe y Ocampo, 2002). Los adultos son conocidos como “San Juanes”, “pololos” o “pololo verde chico”. A. punctatus es una especie limitada a regiones del Sur de Chile (Valdivia – Llanquihue), (Smith, 2002) y su importancia relativa en los ecosistemas es desconocida.

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La larva de H. elegans tiene una abertura anal en forma de C, sin pallidia. El tegillum tiene 59-71 setas curvas. Espiráculos A2-A8 y T son de similar tamaño, espiráculos A1 de tamaño algo más pequeño que los posteriores. El antenito apical tiene una mancha sensorial dorsal y dos manchas sensoriales ventrales. El plato respiratorio del espiráculo toráxico tiene 20-23 aberturas irregulares a través de su diámetro. El área estriduladora maxilar esta formada por una fila de 9-10 dientes agudos, anteriormente dirigidos y recurvados y un tubérculo despuntado distal. El cranium es levemente rugoso y de color rojo. El área estriduladora mandibular es suboval, con aproximadamente 54-62 cordoncillos transversales. El haptomerum tiene 3–4 sensilas, un proceso en forma de pico y ocho setas gruesas en fila transversal.

La larva de A. punctatus tiene una abertura anal en forma de C, sin pallidia. El tegillum tiene 190-200 setas cortas , delgadas y rectas, así también como 2 agrupaciones de setas largas, rectas y delgadas a cada extremo de la abertura anal. Los espiráculos A2-A7 de tamaño similar, A1 de tamaño algo más pequeño que los A2-A7 y A8 más grandes que los anteriores. El antenito apical tiene una mancha sensorial dorsal y dos manchas sensoriales ventrales. El plato respiratorio del espiráculo toráxico tiene 15-18 aberturas irregulares a través de su diámetro. El área estriduladora maxilar esta formada por una fila de 10-12 dientes agudos, anteriormente dirigidos y recurvados y un tubérculo despuntado distal. El cranium rugoso y café rojizo. El area estriduladora mandibular es suboval, con aproximadamente 32-35 cordoncillos transversales. El haptomerum esta desprovisto de sensilas, pero tiene un proceso en forma de pico y ocho setas gruesas en fila transversal.

El género Schizochelus Blanchard esta ampliamente distribuido en la región Neotropical y de acuerdo a Blackwelder, (1944), en Chile esta representado por seis especies: Schizochelus serratus Philippi, Schizochelus breviventris Philippi, Schizochelus longipes Philippi, Schizochelus modestus Philippi, Schizochelus ursulus Philippi, and Schizochelus vestitus Philippi. Dos de las especies, Schizochelus bicoloripes Blanchard and Schizochelus flavescens Blanchard, se encuentran en Brazil. Listados recientes de Evans y Smith, (2009)

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y Mondaca, (2008) indican sólo cinco especies para Chile: S. serratus, S. breviventris, S. modestus, S. ursulus, and S. vestitus. Smith, (2008) y Evans y Smith, (2009) proponen un nuevo ordenamiento en la clasificación de algunos de estos insectos, considerando a S. serratus and S. breviventris como sinonimias de Pristerophora picipennis (Solier), el cual esta presente en Chile y Argentina.

La larva de P. picipennis tiene una abertura anal en forma de Y, sin pallidia. El tegillum con 21-30 setas curvas. Espiráculos A1-A2-A3-A4 y T de similar tamaño, A5-A6 y A7 del mismo tamaño y más pequeños que los anteriores, A8 conspicuamente más pequeño. Antenito apical con una mancha sensorial dorsal y dos manchas sensoriales ventrales. Plato respiratorio del espiráculo toráxico con 9-12 aberturas irregulares a través de su diámetro. Área estriduladota maxilar formada por una fila de 8-12 dientes. Segmentos dorsales del abdomen y lóbulo anal dorsal cubierto con setas delgadas rectas y setas puntiagudas y cortas.

La larva de S. modestus tiene una abertura anal en forma de Y, con 2 pallidia paralelas y longitudinales con 9-10 pali. El tegillum con 13- 16 setas curves. Espiráculo A1 (0,09 mm) levemente más grandes que A2-A8 (0.07mm) de similar tamaño. Plato respiratorio del espiráculo toráxico con 10-12 aberturas regulares a través de su diámetro. Antenito apical con una mancha sensorial dorsal y dos manchas sensoriales ventrales. El área estriduladora maxilar formada por una fila de 8-10 dientes. Segmentos dorsales del abdomen A1–A6 con 3 a 4 filas de setas cortas en forma de hoz y lóbulo anal dorsal cubierto también con setas en forma de hoz.

Los resultados de esta investigación muestra que la taxonomía tradicional nos ha permitido una adecuada separación de las especies en su estado larval. Sin embargo, probablemente en el futuro próximo deban implementarse métodos moleculares que permitan caracterizar a las especies en base a las secuencias de ADN (Dittrich et al., 2006; Pizzo et al., 2006). Esta metodología podría ser importante para separar grupos de gran complejidad como los

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géneros Brachysternus, Sericoides y Aulacopalpus, en los cuales la taxonomía tradicional ha tenido problemas para separar las especies al estado adulto.

Los insectos fitófagos tienen frente a las plantas una serie de desafíos, desde la pobreza nutritiva del recurso planta frente a otros recursos (Blossey & Hunt-Joshi, 2003) como en la necesidad de superar las barreras físicas y químicas que las plantas ponen a los insectos para evitar su destrucción, (Van Der Putten, 2003). Un problema adicional que presentan las plantas es que en los sistemas naturales presentan distribuciones agregadas, frente a ello los insectos fitófagos especialmente los monófagos han debido sufrir un proceso de selección natural de caracteres heredables que les permitan a las hembras responder a estímulos que benefician la sobrevivencia y desarrollo de su descendencia (Thompson and Pellmyr, 1991). Esto se conoce como la hipótesis de la selección por la hembra de sitios de oviposición (preferencia) en los cuales el resultado (performance) es óptimo. Esto se denominado como la hipótesis Preferencia- Performance propuesta por Jaenicke, (1978). Es fácil comprender esta situación tratándose de fitófagos monófagos o con un reducido número de hospederos, pero es más difícil entender en insectos con una amplia polifagia como es el caso de las larvas de escarabaeidos, que se alimentan de las raíces de numerosas plantas y M.O. del suelo. Por ello se estudió este comportamiento en dos especies nativas muy polífagas, determinándose que ambas fueron capaces de seleccionar el sitio de oviposición frente a factores, visuales, físicos y químicos. Tal como en otras especies de escarabaeidos en que el comportamiento de oviposición de la hembra ha sido estudiado (Szendrei e Isaacs, 2005). H. elegans y P. herrmanni respondieron a la presencia de objetos verticales (falsas plantas o plantas), lo cual confirma una respuesta visual, siendo ambas especies capaces de dimensionar la magnitud del estímulo. No obstante, ambas especies también fueron capaces de responder a estímulos químicos producidos por las plantas, concentrando su oviposición en sitios en que ellos estaban presentes. Esto demuestra que ambas especies han experimentado una importante selección natural, para oviponer en sitios con plantas, que son las condiciones que favorecen el potencial biológico (fitness) de la descendencia. Los resultados obtenidos concuerdan completamente con la

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hipótesis Preferencia - Performance propuesta por Jaenike, (1978) y complementan el planteamiento de Scott et al., (2006) sobre el principio de ‘maternidad’.

Tal como era de esperar en insectos de amplia polifagia las señales empleadas para determinar los sitios de oviposición son generales, por lo que es esperable que estas especies puedan colonizar rápidamente nuevos hospederos y convertirse en plagas importantes en estas nuevas introducciones como ha estado sucediendo, pero es altamente improbable que abandonen antiguas plantas hospederas por nuevas.

Un aspecto importante en el manejo cultural de estas especies que se obtuvo producto de esta tesis fue su comportamiento frente a distintos tipos de cubierta. Existieron cubiertas con una escasa atracción como es el caso de aserrín de roble y arena, los cuales podrían ser ocupados en cultivos de bayas. Otros, en cambio, mostraron atracción como es el caso de la paja y en algunos casos del estiércol. El efecto del tiempo de elaboración del estiercol, en la respuesta de atracción por los adultos, sugiere la necesidad de profundizar la investigación en este aspecto antes de aplicar cubiertas como sistemas culturales de manejo.

Los resultados obtenidos con las larvas L1 refutan lo sostenido por numerosos autores que las larvas L1 de los escarabaeidos regularmente no consumen raíces como alimento. De acuerdo a Ritcher, (1958), en la primera fase de colonización y su presencia no tendría mayor relevancia. Esto no es así para las dos especies estudiadas, en las cuales la presencia de raíces es fundamental en las primeras etapas de crecimiento, desarrollo y sobrevivencia, las raíces son relevantes para lograr una buena adecuación biológica o potencial biológico del insecto.

Las respuestas observadas tanto en la selección del sitio de oviposición por las hembras adultas, como en el crecimiento y desarrollo del insecto, confirmarían que los melolontinos

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(P. herrmanni ) requieren en mayor grado del recurso planta que los rutelinos (H. elegans ) y que probablemente los requerimientos nutricionales de los primeros son mayores.

En relación a los tipos de suelos la ausencia de una respuesta clara podría estar dada por la forma como se presentaron los suelos pulverizados, sin estructura y con una compactación uniforme, todo lo cual pudo conspirar para no lograr diferencias. Los resultados obtenidos en el suelo Inceptisol fueron sorprendentes, indicando que la ausencia de altas densidades de estas dos especies en él, se deben a los fenómenos catastróficos que allí ocurren (inundaciones). El mejor desarrollo de las larvas en suelos con niveles de saturación menores de aluminio, es un aspecto de suyo interesante que es necesario abordar.

En resumen, esta tesis abre un plétora de nuevas preguntas, pero al mismo tiempo entrega nuevos conocimientos sobre las características morfológicas del estado larval de diversas especies endémicas, ampliando además la visión para el desarrollo de investigación fundamental y aplicada de este grupo de insectos Además entrega elementos para manejar esta plaga y permite confirmar la hipótesis como selección y perfomance en grupos polífagos y nos permite rechazar afirmaciones sobre los requerimientos nutricionales del primer estadio larval de ambas especies, lo cual constituirá un importante avance en el manejo de estas plagas por ser la forma mas sensible por su pequeño tamaño y capacidad para moverse en el suelo (Suguira et al., 2007)

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ANEXOS

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ANEXO 1: ESTRUCTURAS ESQUEMÁTICAS DE IMPORTANCIA TAXONÓMICA PARA LA DESCRIPCIÓN DE LARVAS DE ESCARABAEIDOS. (A) FORMA LARVAL; (B) CABEZA; (C) CUARTO ANTENITO; (D) EPIFARINGE. Ritcher, (1966).

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ANEXO 2. ESTRUCTURAS ESQUEMÁTICAS DE IMPORTANCIA TAXONÓMICA PARA LA DESCRIPCIÓN DE LARVAS DE ESCARABAEIDOS. (A) MANDÍBULA VISTA DORSAL; (B) MANDÍBULA VISTA LATERAL Y (C) MANDÍBULA VISTA VENTRAL. Ritcher, (1966)

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ANEXO 3. ESTRUCTURAS ESQUEMÁTICAS DE IMPORTANCIA TAXONÓMICA PARA LA DESCRIPCIÓN DE LARVAS DE ESCARABAEIDOS. (A) MAXILA Y LABIO DORSAL; (B) MAXILA LABIO VENTRAL; (C) DIENTES MAXILARES ESTRIDULADORES Y (D) UNCUS DE LA MAXILA. Ritcher, (1966)

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ANEXO 4. ESTRUCTURAS ESQUEMÁTICAS DE IMPORTANCIA TAXONÓMICA PARA LA DESCRIPCIÓN DE LARVAS DE ESCARABAEIDOS. (A) TERGO ABDOMINAL; (B) ESPIRÁCULO TORÁXICO; (C) PATA Y UÑA Y (D) RÁSTER. Ritcher, (1966)

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ANEXO 5. LISTA DE ABREVIATURAS DE ESTRUCTURAS TAXONÓMICAS (Ritcher, 1966; Quintanilla y Fraga, 1969)

A antena LAD lóbulo anal dorsal AA abertura anal LAV lóbulo anal ventral Aa ángulo antenal Lto laeotorma Ac acia Mo molares Aco área cortante MS mancha sensorial Acpa acantoparia Ne nesia Apa acroparia P plegma Asp asperezas Pa palus Ato apotorma Paa paria Bu bulla Pal Palidia BB bárbula Par postartis Br brustia Pco proceso cortador C clipeo Pe pedium Ca campus Pl plegmatium Car cardo Plb palpo maxilar Chpa chaetoparia Ppl proplegmata C1 clitra PrC preclipeo Co corifa Prec precoila Cr crepis PRs plato respiratorio Dst dientes estriduladorea PsC postclipeo Dto dexiotorma Pv proceso ventral E epicráneo S sensilas Ehf esclerito hipofaringeal Sbl setas basolaterales Escr escrobe Scur setas curvas Esp espiráculo sDE setas dorso epicraneales Est estipe Sdm setas dorso molares Eto epitorma SE sutura epicraneal Fo fobae SF sutura frontal Gal galea sFA setas fronto anteriores G1 glosa SFC sutura fronto clipeal Gpa gymnoparia SFP setas fronto posteriores Ha haptomerum Ta tarso He heli Ti tibia Hes hendidura espiracular Tr Trocánter Hla haptolachus U uñas L labro Uc unci Lab Labacoria Un uncus Lac Lacinia Z zigum

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ANEXO 6: DESCRIPCION DE LAS ESTRUCTURAS DE IMPORTANCIA TAXONÓMICA PARA LA DESCRIPCIÓN DE LARVAS DE ESCARABAEIDOS. Ritcher, (1966).

Cabeza. Es hipognata, fuertemente quitinizada y varía considerablemente de color, en general es de forma convexa o subglobosa y comúnmente simétrica, Anexo 1B. En este tagma existen escleritos y regiones de importancia taxonómica, tales como: Frente (F): Esclerito impar situado en la cara anterior de la cabeza entre el epicráneo y el clipeo. Epicráneo (E): Cara dorsal o superior de la cabeza que se extiende desde la frente hasta el cuello. Sutura epicraneal (SE): Sutura en forma de Y , dispuesta sobre la cara dorsal de la cabeza. Sutura frontal (SF): Ramas de la sutura epicraneal que parten de la sutura coronal, y divergen centralmente y anteriormente entre la base de las antenas. Sutura coronal (SC): Rama impar de la sutura epicraneal. Sutura fronto-clipeal (SFC): Sutura que separa a la frente del clipeo. Setas dorso-epicraneales (sDE). Setas fronto-anteriores (sFA). Setas fronto-posteriores (sFP). Clipeo (C): Esclerito impar, ubicado en la cara anterior de la cabeza entre la frente y el labro. También llamado opistoma, dividido en dos porciones, preclipeo (PrC) y postclipeo (PsC). Labro (L) o labio superior: Pieza o lóbulo impar anterior, que articulado al clipeo sirve como cubierta de la cavidad bucal. Ocelos (O): Órgano visual unifacetado. Ojo simple, ubicado en la cabeza sobre o al costado de esclerito antenal basal. Precoila (Prec). Antena. Es cada uno de los dos apéndices sensoriales anillados que se observan en la cabeza. En la mayoría de las subfamilias de los Scarabaeidae, la antena tiene cuatro antenitos sin contar el antenito basal que esta fusionado a la cabeza y no articulado. Los antenitos varían un poco en tamaño en las diferentes especies. El tercer antenito está extendido distalmente, presentando un proceso más o menos obtuso. El cuarto antenito es más corto que el penúltimo y posee frecuentemente manchas sensoriales. Estas manchas sensoriales varían en número en las distintas especies (Hayes, (1929); Ritcher, (1966); Quintanilla y Fraga (1969). Anexo 1C.

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Epifaringe. Es un lóbulo impar adherido a la cara interna del labro. En esta estructura es posible observar varias zonas y estructuras de valor taxonómico, Anexo 1D. Las estructuras según Quintanilla y Fraga, (1969) serían: Acantoparia (Acpa): Porción más externa de la paria portadora de espinas. Acroparia (Apa): Proceso anterior de la paria que generalmente lleva largas setas. Apotorma (Ato): Proceso anterior de la torma y exterior de la fobae. Clitra (Cl): Par de escleritos cortos en el margen anterior de la epifaringe. Corifa (Co): Porción anterior de la epifaringe entre los clitra. Crepis(Cr): Banda esclerosada, delgada y mediana del haptolachus, generalmente asimétrica. Puede estar ausente o reducida a una línea fina. Chaetoparia (Chpa): Parte interna de la paria recubierta por setas de tamaño variado. Dexiotorma (Dto): Torma derecha. Epitorma (Eto): Proyección que se extiende desde el margen interno de la laeotorma pudiendo estar dirigida hacia la parte anterior o posterior de la epifaringe. Epizigum (Ez): Placa o barra esclerosada que se extiende desde el zygum hasta el margen anterior derecho de la epifaringe, justo debajo del clithrum. Fobae (Fo): Grupo de proyecciones frecuentemente esclerosadas que se observan a continuación de la chaetoparia, en el margen interno de la paria. Gymnoparia (Gpa): Parte desnuda de la paria entre la chaetoparia y la acanthoparia. Esta porción de la epifaringe no siempre es distinguible, ya que las setas de la chaetoparia decrecen gradualmente de tamaño y a veces llegan hasta la acanthoparia. Haptolachus (Hla): región media posterior de la epifaringe a continuación del pedium y portadora de los necia, el crepis y gran número de sensilas. Haptomerum (Ha): Región media anterior entre la corypha y el pedium y portadora de los heli, el zigum y varias sensilas. Heli (He): Espinas gruesas y romas que se observan en el haptomerum de la epifaringe. Laeotorma (Lto): Torma izquierda. Nesium (nesia) (Ne): Uno o dos escleritos ubicados entre los extremos internos de las tormas y anteriormente al crepis. Plegma (P): Cada uno de los pliegues del plegmatum. Plegmatum (Pl): Región lateral de la epifaringe portadora de pliegues o plegmatas y marginada por la acantoparia. Paria (Pa): Región lateral par de la epifaringe que se extiende desde el clithrum, epizigum y haptomerum (o en su lugar el tylus) hasta las tormae, delimitándose del pedium por una serie de setas de la chaetoparia y posteriormente por las phobae. Pedium (Pe): Región central de la epifaringe entre el haptomerum, las parias y el

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haptolachus. Proplegmatium (Ppl): Región par submarginal y anterior de la epifaringe portadora de pliegues. Pternotorma (Pto): Proceso curvado posterior de la laeotorma, a veces también presente en la dexiotorma. Sensila (S): Complejo estereoreceptor constituido por el tegumento. Una célula sensorial y células asociadas. Órgano de los sentidos. Tilus (T): Porción esclerosada en la parte anterior de la epifaringe que cubre total o parcialmente los elementos del epizigum, corifa y haptomerum fusionado. Zigum (Z): Esclerito del haptomerum en forma de arco o alargado, ubicado anteriormente a los heli.

Mandíbulas. Es el primer par de apéndices del aparato bucal de aspecto y tamaño variable. Son fuertemente quitinizadas y algunas más largas que anchas. El área distal es mucho más oscura que la proximal, la cual generalmente es del mismo color que la cabeza. En algunas especies existen diferencias entre la mandíbula derecha e izquierda. Las mandíbulas exhiben dos regiones, una proximal o área molar y una región distal o cortante. El área molar de la mandíbula izquierda como regla es más larga que la derecha, lateralmente la mandíbula presenta una superficie aplastada, la que lleva unas pocas setas, Anexo 2 A y B. La superficie ventral de las mandíbulas posee en muchas especies un área estriada transversal de forma oval, que constituye el área estriduladora (Ast), Anexo 2 C. Otras zonas y estructuras de interés taxonómico en las mandíbulas son: carina dorsal (Cd); asperezas (Asp); acia (Ac): que sólo esta presente en la mandíbula derecha y cubre la epifaringe cuando la mandíbula está cerrada, jugando un rol importante en la molienda del alimento. Proceso ventral (Pv); escrobe (Esc); área cortante (Aco); brustia (Br); calx (Cal); postartis (Par); preartis (Prar); setas basolaterales (Sbl); setas dorso-molares (Sdm) y molares (Mo).

Maxilas. Es el segundo par de apéndices del aparato bucal, presenta el usual número de partes que encontramos en el aparato tipo masticador, excepto que la galea y lacinia pueden o no estar fusionadas para formar una estructura llamada mala (Ma). La lacinia (Lac), galea (Gal), cardo (Car) y estipe (Est), son partes en que se divide la maxila, Anexo 3 AyB.

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La lacinia y galea, llevan apicalmente setas fuertemente esclerozadas llamadas unci (Uc), (pl. uncus) (Un), Anexo 3 D. Otros escleritos y estructuras presentes en las maxilas son los palpos maxilares (Pmx) y los dientes estriduladotes (Dst), Anexo 3 C.

Labio. Pieza bucal posterior, mediana y compleja, proveniente de la fusión del segundo par de maxilas. En el se pueden observar: palpos labiales (Plb); mentum (Me); prementum (PreMe); postmentum (PsMe); glosa (Gl); esclerito hipofaringeal (EhF); fosa hipofaringeal (FhF); lóbulo lateral (Ll) y proceso cortador (PCo), Anexo 3 A y B. Tórax. Esta región esta constituida por tres segmentos las cuales llevan cada una un par de patas y el segmento protoráxico un par de espiráculos. El protórax puede o no estar dividido dorsalmente en dos o tres subsegmentos. El meso y metatórax posee tres escleritos, el preescudo (PrSC), escudo (SC) y escutelo (SCL), además se puede encontrar otras regiones como : subescudo (SES), postescutelo (PSCL), lóbulo pleural (LPL), área espiracular (ASp), eusterno (Eus), area pedal (Apd) y esternelo (EsT), los cuales pueden llevar pequeñas y largas setas.

Patas. Cada pata posee una coxa (Cx) convexa, trocánter (Tr), fémur (Fe), tibia (Ti), tarso (Ta) y una uña (U), la cual lleva generalmente dos setas, Anexo 4 C.

Espiráculos toráxicos. En ellos existen regiones tales como plato respiratorio (PRs), hendidura espiracular (HEs) y la bulla (Bu). En el plato respiratorio existen perforaciones, las cuales varían entre géneros y especies en cuanto al número y forma. La concavidad espiracular esta dirigida hacia la región posterior del cuerpo, Anexo 4 B.

Abdomen. Este tagma presenta diez segmentos, los que están divididos generalmente por una serie de grandes y profundos pliegues, formando subsegmentos. Existen tres subsegmentos en la mayoría de los segmentos, aunque en algunos de ellos no son iguales o están ausentes, Anexo 4 A.

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Espiráculos abdominales: El tamaño de ellos varía a veces de segmento en segmento. Poseen las mismas regiones de los espiráculos toráxicos. La concavidad espiracular esta dirigida anteriormente y el número de espiráculos es ocho.

Ráster: Es un área setosa localizada centralmente en el último segmento abdominal. En esta región se encuentran una serie de estructuras y conformaciones setosas que sirven como características taxonómicas, ellas son: lóbulo anal dorsal (LAD), lóbulo anal ventral (LAV), abertura anal (AA), esta difiere en forma, en los distintos grupos, pudiendo ser obtuso, transversalmente curvada o en forma de Y. También se encuentran los palus (Pa), que son filas de setas dispuestos en forma paralela o perpendicular a la abertura anal. La séptula (Sep), es un área desnuda entre las palidias, el tegillum (Teg), región lateral a las palidias conformadas por setas curvas (scur), los teges (te), setas cortas y derechas , ubicadas en la región anterior a la abertura anal. Campus (ca) zona anterior del segmento abdominal. Bárbula (BB), setas largas, ubicadas latero-ventralmente en el último tercio del segmento abdominal, Anexo 4 D.

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