Morphology of the European species of the aphid genus Eulachnus (Hemiptera: Aphididae: Lachninae) - A SEM comparative and integrative study.

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Morphology of the European species of the aphid genus Eulachnus (Hemiptera: Aphididae: Lachninae) – A SEM comparative and integrative study

Mariusz Kanturski a,∗ , Jagna Karcz b,c , Karina Wieczorek a a

Department of Zoology, Bankowa 9, 40-007 Katowice, Poland Scanning Electron Microscopy Laboratory, Jagiello´ nska 28, 40-032 Katowice, Poland Q2 c Faculty for Biology and Environmental Protection, University of Silesia, Katowice, Poland

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a r t i c l e

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i n f o

a b s t r a c t

Article history: Received 15 March 2015 Received in revised form 14 April 2015 Accepted 5 May 2015 Available online xxx

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Keywords: Aphids Chaetotaxy CPD Cryo-SEM HMDS Pine-pest SEM

1. Introduction

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Scanning electron microscopy (SEM) methods were used for the ﬁrst time to elucidate the external morphology of the European species of the genus Eulachnus (Hemiptera: Aphididae: Lachninae), a representative genus of the conifer-feeding aphids tribe Eulachnini. We examined and compared the external morphology of apterous and alate viviparous females from the parthenogenetic generation as well as oviparous females and alate males belonging to the sexual generation. FE-SEM images based on HMDS and cryo-SEM preparation techniques revealed better image quality than the CPD technique in regard to surface tension and morphological signs of cell deteriorations (i.e., existence of depressions, drying artifacts and membrane blebs). Three morphologically different species groups “agilis”, “brevipilosus” and “cembrae” were proposed due to the differences in head, antennae, legs and dorsal chaetotaxy as well as dorsal sclerotization. The most characteristic features and differences of representatives of these groups are presented and discussed. © 2015 Published by Elsevier Ltd.

The palaearctic genus Eulachnus Del Guercio, 1909 comprises about 12–18 species, the most well studied of which are from Europe (Blackman and Eastop, 1994, 2015; Kanturski and Wieczorek, 2014a). Aphids from this genus are recognized as pine pests and may cause damage in forests, nurseries and ornamental trees and shrubs (Heinze, 1962; Bliss and Kearby, 1971; Bliss et al., 1973; Carter and Maslen, 1982; Zondag, 1983; Binazzi, 1989; Alford, 2012). Eulachnus species are characterized by a small, narrow, spindle-shaped body and a hidden and timid mode of life. Unlike other aphids, species from this genus do not form large and numerous colonies, feeding mainly separately between pine needles (Binazzi and Scheurer, 2009; Kanturski and Wieczorek, 2014a). The genus Eulachnus is a member of the important conifer-feeding aphids tribe Eulachnini Baker, 1920, which includes morphologically related, narrow-bodied species from the genus Essigella Del Guercio, 1909 from North America and a monotypic genus Pseudessigella Hille Ris Lambers, 1966 from Pakistan. Taxa from the genera Cinara Curtis, 1835 and Schizolachnus Mordvilko, 1909

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∗ Corresponding author. Tel.: +48 509816296. E-mail address: [emailprotected] (M. Kanturski).

(Favret, 2015) also belong to the tribe Eulachnini. They are characterized by an oval or rounded body shape and they tend to often form large colonies and thus conform with other genera from the subfamily Lachninae Herrich-Schaeffer, 1854 and most Aphididae Latreille, 1802 species (Blackman and Eastop, 1994, 2015; Heie, 1995). Eulachnus representatives are characterized by 6-segmented antennae and a short, blunt apical rostrum segment without accessory setae, which differentiates Eulachnus from Cinara and Schizolachnus in the European aphid fauna. Apterous (wingless) viviparous females are most common, whereas other morphs such as alate (winged) viviparous females and the sexual generation (apterous oviparous females and alate males) are rare and, in some species, still remain unknown. Species belonging to the genus Eulachnus are treated as morphologically similar and only Binazzi (1983) and Mier-Durante and Binazzi (1997) have used the term “agilis” group for species which were similar to E. agilis (Kaltenbach, 1843) – i.e. E. rileyi (Williams, 1911), E. tuberculostemmatus (Theobald, 1915), E. mediterraneus Binazzi (1983) and E. intermedius Binazzi (1989). No other authors have ever commented on the term “agilis” group. However, a revision of the European species of the genus Eulachnus reveals that, in addition to the “agilis” group, there are two other distinctive groups constituting the genus – the “brevipilosus” and the “cembrae” groups (Kanturski et al., in preparation).

Please cite this article in press as: Kanturski, M., et al., Morphology of the European species of the aphid genus Eulachnus (Hemiptera: Aphididae: Lachninae) – A SEM comparative and integrative study. Micron (2015), http://dx.doi.org/10.1016/j.micron.2015.05.004

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Because of their hidden mode of life and similarities in morphological characters, representatives from the genus Eulachnus are considered as difﬁcult to determine. As the light microscopy can be an insufﬁcient method in the identiﬁcation of those aphids, further morphological research using scanning electron microscopy (SEM) is needed – as well as to conﬁrm the existence of species groups (“agilis”, “brevipilosus” and “cembrae”) within this genus. It is well known that preparation of soft and delicate biological (plant and animal) material for SEM by chemical ﬁxation and critical point drying (CPD) results in shrinkage of tissues (Bray et al., 1993; Braet et al., 1997; Araujo et al., 2003; Talbot and White, 2013). Therefore, in this paper we evaluate three biological sample-preparation methodologies for imaging the morphology of surface structures of aphids, with the representatives of the genus Eulachnus as an example. The preparation methods examined are: ethanol ﬁxation with critical-point drying (CPD), ethanol ﬁxation followed by hexamethyldisilazane (HMDS) drying and direct imaging by cryo-SEM (Kashi et al., 2014). Preparation methods are evaluated based on the resulting image quality and reduced potential for artifacts. Due to the poor state of knowledge and the difﬁculties with the determination of Eulachnus species, as well as their importance as pine pests, a comprehensive and comparative study of the morphology of all morphs is provided for the ﬁrst time and the results are presented in this paper.

2. Materials and methods

2.1. Taxon sampling

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Regular ﬁeld studies and collecting of the representatives of the genus Eulachnus were carried out in Europe (Poland, Austria, Czech Republic, Denmark, England) in the 2011–2014 seasons. The aphids were collected from May to November, directly from pine needles or shaken out onto an entomological umbrella. The goal was to collect all morphs in Poland: E. agilis, E. rileyi (Katowice, ´ askie, ˛ Lachowice), E. brevipilosus Börner, 1940 (BranOborniki Sl ice, Katowice), E. nigricola (Paˇsek, 1953) (Lachowice, Katowice);

Denmark and England: E. agilis, E. brevipilosus (Copenhagen, London); and Austria: E. agilis, E. cembrae Börner, 1950 (Malnitz), E. rileyi (Graz). One species, E. tuberculostemmatus, also found in the Mediterranean area, was collected in July 2014 in Tajikistan (Tigrova Balka). All examined adult individuals were preserved in 70% ethanol and then used for observation in light and scanning electron microscopy. We examined 43 Eulachnus specimens from all three proposed morphological groups of species. The collection data is summarized in Table 1. The genitalia terminology is given after Wieczorek et al. (2012). 2.2. Light and scanning electron microscopy (SEM) Mounting for light microscopy. Field-collected specimens were preserved in 70% ethanol and prepared in KOH, chloralphenole and chloralhydrate water bath and ﬁnally ﬁxed in Faure–Berlese ﬂuid (Kanturski and Wieczorek, 2012). The specimens were examined using a Nikon Ni-U light microscope and were photographed with a Nikon DS-Fi2 camera. For SEM, adult female and male individuals were ﬁxed and stored in 70% ethanol and then three different methods of sample preparation were used: (1) ethanol dehydration followed by critical point drying (CPD); (2) ethanol dehydration and hexamethyldisilazane (HMDS) drying; (3) direct imaging by cryo-SEM. After 70% ethanol ﬁxation, CPD and HMDS aphid material was dehydrated in a graded ethanol/water series of 75%, 80%, 90%, 95%, and 100% for 10 min in each concentration with three 100% ethanol changes. Dehydrated samples were subsequently critical-point dried using carbon dioxide in a Pelco CPD2 apparatus (Ted Pella Inc., Redding, CA, USA), or treated (3 × 10 min) with HMDS (Polysciences Inc., USA). After the third change, specimens remained in HMDS until all of the solution evaporated and were ﬁnally air dried in a desiccator). For both methods, samples were mounted on aluminum stubs with double-sided adhesive carbon tape and sputter-coated in a Pelco SC-6 sputter coater (Ted Pella Inc., Redding, CA, USA) with a thin ﬁlm of gold to improve the electrical conductivity of the sample surface. After processing, samples were imaged by the

Table 1 Examined species and morphs of the European representatives of the genus Eulachnus. Material examined Morph E. agilis 1 apterous viviparous female 1 apterous viviparous female 1 apterous viviparous female 1 alate viviparous female 1 alate viviparous female 3 oviparous female, 3 alate males, E. rileyi 1 apterous viviparous female 2 apterous viviparous females 1 alate viviparous female 1 alate viviparous female 3 oviparous females E. brevipilosus 1 apterous viviparous female 1 apterous viviparous female 2 alate viviparous females 1 alate viviparous female 3 oviparous females E. nigricola 1 apterous viviparous female 2 apterous viviparous females 3 oviparous females, 1 alate male E. tuberculostemmatus 3 apterous viviparous females E. cembrae 3 apterous viviparous females

Locality

Host plant

Date

Determination data

Katowice, Poland London, England Copenhagen, Denmark ´ askie, ˛ Oborniki Sl Poland Malnitz, Austria Katowice, Poland

Pinus silvestris P. silvestris P. silvestris P. silvestris P. mugo P. silvestris

24.06.2011 17.09.2013 27.05.2012 14.07.2013 20.07.2014 14. 10. 2014

leg. M. Kanturski, Silesia University, no. 06/11/10 leg. M. Kanturski, Silesia University, no. 09/13/56 leg. M. Kanturski, Silesia University, no. 05/12/20 leg. M. Kanturski, Silesia University, no. 07/13/13 leg. K. Wieczorek, Silesia University, no. 07/14/18 leg. M. Kanturski, Silesia University, no. 10/14/6

´ askie, ˛ Oborniki Sl Poland Katowice, Poland Lachowice, Poland Graz, Austria Katowice, Poland

P. nigra P. nigra P. nigra P. nigra P. nigra

14.07.2011 24.06.2013 05.05.2012 20.06.2012 14.10.2014

leg. M. Kanturski, Silesia University, no. 07/11/15 leg. M. Kanturski, Silesia University, no. 06/13/12 leg. M. Kanturski, Silesia University, no. 05/12/3 leg. M. Kanturski, Silesia University, no. 06/12/15 leg. M. Kanturski, Silesia University, no. 10/14/7

Copenhagen, Denmark London, England Branice, Poland Katowice, Poland Branice, Poland

P. silvestris P. silvestris P. mugo P. silvestris P. mugo

27.05.2012 17.09.2013 01.06.2014 05.06.2014 01.11.2014

leg. M. Kanturski, Silesia University, no. 05/12/21 leg. M. Kanturski, Silesia University, no. 09/13/55 leg. M. Kanturski, Silesia University, no. 06/14/2 leg. M. Kanturski, Silesia University, no. 06/14/5 leg. M. Kanturski, Silesia University, no. 11/14/1

Lachowice, Poland Katowice, Poland Katowice, Poland

P. nigra P. nigra P. nigra

05.05.2012 14. 10. 2014 31.10.2014

leg. M. Kanturski, Silesia University, no. 05/12/4 leg. M. Kanturski, Silesia University, no. 10/14/8 leg. M. Kanturski, Silesia University, no. 10/14/20

Tigrova Balka, Tajikistan

P. pinea

20.07.2014

leg. M. Walczak, Silesia University, no. 07/14/TMK5

Malnitz, Austria

P. cembra

20.07.2014

leg. K. Wieczorek, Silesia University, no. 07/14/20

103 104 105 106 107 108 109 110 111 112

113

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Fig. 1. Scanning electron micrographs showing apterous viviparous females of the species groups of the genus Eulachnus: dorsal view: E. agilis (a) (HMDS), (b) E. brevipilosus (CRYO), (c) E. cembrae (CPD); lateral view: (d) E. agilis (CPD), (e) E. brevipilosus (HMDS), (f) E. cembrae (HMDS); dorsal and lateral side of head and pronotum: (g) E. agilis (CPD), (h) E. brevipilosus (HMDS), (i) E. cembrae (HMDS).

Fig. 2. Light microscope images showing apterous viviparous females of the species groups of the genus Eulachnus. (a) E. rileyi, (b) E. brevipilosus with well visible blunt ARS (black arrow), (c) E. cembrae.

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Hitachi SU8010 ﬁeld emission scanning electron microscope FESEM (Hitachi High-Technologies Corporation, Tokyo, Japan) at 5, 10 and 15 kV accelerating voltage with a secondary electron detector (ESD) and at the working distance (WD) of 10–30 mm. For cryo-SEM imaging, 70% ethanol ﬁxed adults were mounted on aluminum stubs with a layer of carbon conductive glue (1:1, Tissue Tek O.C.T. compound, colloidal graphite from Agar Scientiﬁc) on the cold copper holder. The sample holder was attached to a transfer rod and immediately frozen (−196◦ C) in liquid nitrogen slush using a Quorum PP 2000 cryo-preparation stage (Polaron, Quorum Technologies, United Kingdom). The holder with the frozen samples was raised and cryo-transferred at the temperature of liquid nitrogen vapors to the chamber of the cryo-unit where the sample was subjected to sublimation at −70◦ C for 10 min until all visible ice crystals disappeared. The samples were then sputter coated with platinum (5 nm thickness). Following the coating, the specimens were transferred to the cooling stage of the Hitachi SU8010 FESEM and imaged at −130◦ C, at an acceleration voltage of 5–10 kV and at a WD of 10–30 mm.

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3. Results

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The FE-SEM images from aphids sample preparation based on CPD, HMDS and cryo-SEM techniques were compared in different magniﬁcations as presented in Figs. 1–14. Compared 160 to the FE-SEM images based on the CPD technique (Fig. 1c), 161 HMDS and cryo-SEM images have well-preserved outline surface 162 structures (Fig. 1a and b) and details of surface morphol163 ogy are clearly visible. Visually, the HMDS and cryo-SEM 164 techniques have preserved surface morphology better without 165 wrinkles and collapsed areas than the CPD technique could 166 do as described in images with 30–200× magniﬁcation levels 167 Q5 (Figs. 1c, d, g; 3i and j; 4b and e; 5e; 6d; 7a–d; 9; 10a–d; 11a–c; 13a). 168 The drying collapsed artifacts were present in larger quanti169 ties on the specimens prepared in CPD technique as compared 170 with the specimens in the HMDS and cryo-SEM techniques 171 (Figs. 1a, b, e, f, h, i; 3a–h; 4a, c, d; 5a–d; 6a–d; 7e–h; 8; 10e–h; 11d–f; 172 12; 13b and c; 14). In the collected and prepared insect speci173 mens, a different type of maintenance of the material was observed. 174 Abdominal and thorax structures were better preserved in samples 175 collected in the spring and in the autumn – as well as the head and 176 thorax structures of the alate viviparous females and alate males. 177 158 159

178

179 180 181 182 183 184 185 186 187 188 189 190 191 192 193

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3.1. Apterous viviparous females Aphids of the genus Eulachnus are characterized by a very delicate body. The cuticle is thin and mostly membranous, especially on the abdomen, with long setae arising from distinct and visible scleroites, which can fuse into larger sclerites in some species from the “agilis” group. Species of the “brevipilosus” group differ from the former by very short and inconspicuous setae arising from small and separate scleroites. The most differing species from the “agilis” group belong to the “cembrae” group, which is characterized by the absence of any dorsal scleroites or sclerites and the short and few setae arising from the cuticle surface (Figs. 1 and 2). Those characters explain why in the “brevipilosus” and “cembrae” groups, the setae on the dorsum are almost invisible in life and even in magniﬁcation c.a. 40–60× using light microscopy (Fig. 2). Long and often rigid body setae of the “agilis” group species are evident and can be seen even at lower magniﬁcation. 3.1.1. Head morphology and chaetotaxy The head of the representatives of all species groups of Eulachnus is small, but the posterior margin is well marked. On both sides of the head there are big and well developed compound eyes with residual, almost invisible triommatidia. The triommatidia are

Fig. 3. SEM images of dorsal side of head and pronotum of apterous viviparous females of the genus Eulachnus. (a) Head and pronotum of E. agilis (HMDS), (b) frontal setae of E. agilis (HMDS), (c) head and pronotum of E. tuberculostemmatus (CRYO), (d) frontal setae of E. tuberculostemmatus (CRYO), (e) head and pronotum of E. brevipilosus (HMDS), (f) frontal setae of E. brevipilosus (HMDS), (g) head and pronotum of E. nigricola (HMDS), (h) frontal setae of E. nigricola (HMDS), (i) head and pronotum of E. cembrae (CPD), (j) frontal setae of E. cembrae (CPD). h – head, pr – pronotum, fr – frons, v – vertex.

present but weakly developed from the compound eyes by the absence of the ocular tubercle as in most aphids. They are present as 3 bigger ommatidia surrounded by smaller ommatidia of the compound eyes. Setae on the head (and pronotum) are long (up to 0.150 mm long), ﬁne or rigid with more or less pointed apices in E. agilis (Fig. 3a and b) and shorter (not longer than 0.085 mm long), with slightly capitate apices in E. tuberculostemmatus (Fig. 3c

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Fig. 4. SEM of antennal segment III chaetotaxy of apterous viviparous females. (a) E. agilis (HMDS), (b) E. rileyi (CPD), (c) E. tuberculostemmatus (CRYO), (d) E. brevipilosus (HMDS), (e) E. cembrae (CPD).

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and d) – “agilis” group. Head setae in the “brevipilosus” (Fig. 3e–h) and “cembrae” groups can be divided into short (up to 0.070 mm long) or very short (not longer than 0.057 mm long) on the dorsal side and much longer (about 0.080 mm long) on the front and the ventral side of the head with big capitate apices (Fig. 3i and j). The chaetotaxy on antennal segment III is speciﬁc for each of the species groups. In the “agilis” group, setae are the longest, sometimes thick and stiff with pointed apices in E. agilis and E. rileyi (Figs. 1a, 2a, 4a and b) and much shorter with blunt and pointed setae in E. tuberculostemmatus (Fig. 4c). On the contrary, setae of the representatives of the “brevipilosus” (Fig. 4d) and “cembrae” (Fig. 4e) groups are delicate, very short with blunt and slightly capitate apices, and never longer than the diameter of the segment. Ventrally, the clypeus is well developed; the labrum is short and small, rostrum with the longest third segment. The shape of the ARS is one of the most characteristic features of Eulachnus with 6-segmented antennae Eulachnini (Cinara, Eulachnus, Schizolachnus). The IV segment of the ARS (the last, apical rostrum segment) is short and blunt with an extremely short, button-shaped, dark and strongly sclerotized apical part, treated by some authors as a

Fig. 5. SEM of hind tibiae of apterous viviparous females. (a) E. rileyi (HMDS), (b) E. tuberculostemmatus (CRYO), (c) E. brevipilosus (HMDS), (d) E. nigricola (HMDS), (e) E. cembrae (CPD).

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Fig. 6. SEM of dorsal side of abdominal segments III of apterous viviparous females. (a) E. agilis (HMDS), (b) E. tuberculostemmatus (CRYO), (c) E. brevipilosus with very short setae (black arrows, HMDS), (d) E. cembrae with a few setae not arising from scleroites (white arrows, CPD). sc – scleroites at setae bases.

Fig. 7. SEM of alate viviparous females of the genus Eulachnus. E. rileyi (CPD): (a) head and pronotum, (b) antenna, (c) antennal segment III chaetotaxy, (d) fore wing; E. brevipilosus (HMDS): (e) head and pronotum, (f) antenna, (g) antennal segment III chaetotaxy, (h) fore wing. h – head, pr – pronotum, fr – frons, v – vertex, oc – ocellus (III–VI) antennal segments III–VI.

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Fig. 8. SEM of fore leg complex of sensory structure of alate viviparous female of E. brevipilosus. (a) Membranous area between trochanter (tr) and femur (f) with pillow-shaped structure (white arrow), (b) location of sensilla of the surface of the structure, (c) sensilla, (d) sensilla and rufﬂed surface of the structure (HMDS).

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246 247 248

V segment (Fig. 2b and c). None of the European representatives of the genus have accessory setae on the ARS, which is another feature characteristic only for Eulachnus within Eulachnini and Lachninae as a whole in Europe. 3.1.2. Hind tibiae chaetotaxy Different types of setae on hind tibiae have great signiﬁcance in the determination of the species in the genus Eulachnus. The hind tibiae chaetotaxy demonstrates the general trends distinguishing the species and species groups. Species belonging to the “agilis” group, in this section represented by E. rileyi and E. tuberculostemmatus, are characterized by long and rigid setae with pointed or slightly blunt apices, especially on the outer side of tibiae (Fig. 5a and b). Outer tibiae setae in the “brevipilosus” group are always capitate, but of different length: very short in E. brevipilosus (Fig. 5c) and always longer for that diameter of tibiae in E. nigricola (Fig. 5d), while the inner setae are always pointed and of variable length. Outer setae of species from both groups are also much longer and are arranged at a greater angle, while the hind tibiae setae of E. cembrae are very short, slightly capitate and almost “lying” on the surface of the tibiae (Fig. 5e). 3.1.3. Dorsal side of the body – sclerotization and chaetotaxy The dorsal side of the body represents the biggest differences among the species groups. The dorsum of the species from the

Fig. 9. SEM of fore leg complex of sensory structure of alate viviparous female of E. rileyi. (a) Membranous area between trochanter (tr) and femur (f) with smooth structure (white arrow), (b) outer side of structure with sensilla (dotted arrow), (c) surface of structure with mushroom-shaped sensilla (CPD).

“agilis” and “brevipilosus” groups is covered by small and generally oval or irregular scleroites at setae bases. In the “agilis” group, the dorsum is covered by very distinct long setae, which can be spindle-shaped and rigid as in E. agilis (Fig. 6a) or short like in E. tuberculostemmatus (Fig. 6b), however always longer than the diameter of the scleroites. Apices of setae in this group are also always pointed. The dorsum of species belonging to the “brevipilosus” group is characterized by poorly visible scleroites, probably due to the thick layer of wax, which covers the scleroites as well. The setae are very short, slightly pointed or blunt and never longer than the diameter of the scleroite (Fig. 6c). E. cembrae is the most signiﬁcant species due to the absence of dorsal abdominal scleroites. The setae are very few, very short and inconspicuous, with capitate apices. At the setae base there is only a small circular or oval ring (Fig. 6d).

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Like in most Aphididae species with alate males, they are often very similar to the alate viviparous females except for numerous secondary rhinaria on antennal segments III–VI and sclerotized genitalia consisting of a pair of parameres, basal part of phallus and membranous aedeagus. The alate males of the European species are also similar in general morphology to the alate viviparous females and manifest the “agilis” and “brevipilosus” groups characters, especially in the length and shape of setae. Males of E. agilis are characterized by very long, ﬁne but rigid setae with pointed apices on the head, antennae, pronotum and legs (Fig. 10a–d), while the male of E. nigricola is the smallest of all known Eulachnus males with chaetotaxy described in alate viviparous female of “brevipilosus” group – longer setae on the frons and shorter and much delicate on the vertex and pronotum. Also the setae on antennae are much shorter than in E. agilis, but also ﬁne and pointed (Fig. 10e–g). Hind legs have also shorter and rigid setae with capitate apices (Fig. 10h). Antennae of the males are covered by numerous small and rounded rhinaria lying on the whole surface of the segments

Slightly capitate Capitate Capitate Blunt

Longer Longer

Pointed and blunt

As long as or longer

Blunt

Pointed or slightly blunt As long as or loner Blunt Pointed or blunt

Blunt – Pointed Shorter Blunt Shorter Blunt Longer Pointed As long as or longer

Blunt Blunt Slightly capitate Blunt

Absent

Slightly capitate Shorter than BD III Capitate or blunt Shorter than BD III Capitate or blunt Shorter than BD III

E. nigricola

Present Present

Pointed or slightly blunt Shorter Longer

Blunt Slightly capitate

305

Pointed

304

Pointed

303

Longer

302

Longer

301

Longer

300

Blunt

299

Pointed or blunt

298

Pointed

297

Pointed or blunt

296

Blunt As long as or longer

295

Blunt Shorter and longer

294

Pointed Longer

293

Pointed Longer

292

Slightly blunt

291

Pointed

290

Pointed

289

Blunt Longer than BD III

288

Pointed or blunt As long as or longer than BD III Slightly blunt

287

Blunt As long as or longer than BD III Blunt

286

Slightly blunt As long as BD III

285

Pointed Longer than BD III

284

Pointed or blunt Longer than BD III

283

Present

282

Present

281

Present

280

Present

279

Present

278

Present

277

Dorsal sclerotization Head setae shape Antennal setae length Antennal setae shape Thorax setae Abdominal setae length to the width of scleroite Abdominal setae shape Tibiae setae length to the width of tibiae Tibiae setae shape

276

E. brevipilosus

275

“brevipilosus” group

274

E. intermedius

273

E. mediterraneus

272

E. alticola

271

E. tuberculostemmatus

270

E. rileyi

269

E. agilis

3.3. Alate males of the genus Eulachnus

268

“agilis” group

307

267

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Alate morphs of species from the “agilis” and “brevipilosus” groups are characterized by a large head with big compound eyes and three ocelli and a much more expanded thorax due to the presence of wings. As in apterous viviparous females, the main differences between those groups are generally manifested in the type of chaetotaxy. The alate of the “agilis” group are characterized by the longest and pointed setae – especially on the head, antennae and pronotum (Fig. 7a–c), while the species from the “brevipilosus” group have two types of setae on the head: longer with capitate apices on the frons and very short and pointed on the vertex (Fig. 7e). The very short setae are almost invisible on the antennae (Fig. 7f). Antennal segment III of species of the “brevipilosus” group may sometimes have up to 3 secondary rhinaria, which is an exceptional case within all European species of Eulachnus. Also the pronotum in the “agilis” and “brevipilosus” group shows several differences, mainly in the shape of the posterior border which in E. rileyi is clearly arched, whereas in E. brevipilosus it is ﬂat. The fore wings of all studied species of Eulachnus are characterized by a weakly developed once-branched media. Wings of the species from the “agilis” group are also longer and wider than the wings of the “brevipilosus” group and have less spicules on the membrane (Fig. 7d and h). The chaetoraxy of hind tibiae is very similar to that in apterous viviparous females. During the analysis of the alate viviparous females of E. rileyi and E. brevipilosus, a hitherto unknown complex of sensory structures with numerous sensilla in this group of insects has been found (Figs. 8 and 9). The complex occurs on the fore leg on the membranous area between trochanter and femur. In the alate of E. brevipilosus, the complex is pillow-shaped with numerous peg-like sensilla (Fig. 8a and b). The surface of the structure is strongly rufﬂed and the cone or tusk-shaped sensilla lye in circular cavities with well-developed edges (Fig. 8c and d). Between the cones and cavity edges, there is also a rufﬂed or smooth area visible. Most probably due to this construction, the sensilla can be ﬂexible. The same area on the fore leg in E. rileyi bears most probably a hom*ologous, smooth, pillow-shaped structure without the sensilla described above (Fig. 9a). Only on the outer surface of the structure are there about 2–3 cavities with small, inverted mushroom-shaped sensilla present (Fig. 9b and c). Like in E. brevipilosus, observations of middle and hind leg did not reveal any structures and it is most likely that such structures were not previously reported in Lachninae.

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3.2. Alate viviparous females general body morphology and chaetotaxy

Table 2 Summarized general characters of sclerotization and chaetotaxy existing in all known morphs of the European species of the genus Eulachnus. BD III – basal articular diameter of antennal segment III.

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Fig. 10. SEM of alate males of the genus Eulachnus. E. agilis (CPD): (a) head and pronotum, (b) antennal segment III with secondary rhinaria, (c) the shape and location of secondary rhinaria, (d) hind tibia chaetotaxy; E. nigricola (HMDS): (e) head and pronotum, (f) antennal segment III with secondary rhinaria, (g) the shape and location of secondary rhinaria, (h) hind tibia chaetotaxy. h – head, pr – pronotum, fr – frons, v – vertex, oc – ocellus.

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in E. agilis (Fig. 10b and c) and several to over a dozen rhinaria in E. nigricola placed in 1–2 rows on the segments (Fig. 10f and g). 3.3.1. The genitalia of Eulachnus males In E. agilis, the parameres are located above the basal part of the phallus, clearly visible and basally fused. The lobate parts of parameres arise into a distinct, ﬁnger-like projection toward the base of the phallus (Fig. 11a). The parameres bear numerous, long setae on the entire surface with the exception of a few, short setae distributed on the projections (Fig. 11b). The basal part of the phallus is short, ﬂattened and smooth, with a few short, rigid and pointed setae (Fig. 11c). The aedeagus is poorly visible. The genitalia of E. nigricola are in general similar to those of E. agilis, but the parameres have shorter setae and the apex of the basal part of phallus is clearly circular without any setae and has a rough surface (Fig. 11d–f). The surface of the basal part of the phallus of E. nigricola has an extraordinary surface covered by numerous spicule-like projections of variable shape (Fig. 12a–c). Near the paramere base, many very small sensilla gathered in small groups were found (Fig. 12d). The structures have a circular and rufﬂed ﬂange with a thick and rigid projection with either a pointed or rounded apex (Fig. 12e and f).

3.4. Oviparous females The oviparous females have a very similar morphology to the apterous viviparous females. In the case of Eulachnus, the oviparae manifest most of viviparae characters, especially regarding body chaetotaxy and sclerotization. There are two main morphological characters distinguishing them from the parthenogenetic morphs: the pseudosensoria on slightly swollen hind tibiae and a wider and much more hairy genital plate. The pseudosensoria of the studied “agilis” and “brevipilosus” group species are developed as small, circular or oval low protuberances with a grainy surface (Fig. 13a and b) and with little contraction in the center in some species (Fig. 13c). The genital plate is also often longer and bigger with many long setae (even in the “brevipilosus” group) (Fig. 14).

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Fig. 11. External genitalia of males of the genus Eulachnus. E. agilis (CPD): (a) genitalia – general view, (b) paramere-p, (c) basal part of phallus-bp; E. nigricola (HMDS): (d) genitalia – general view, (e) paramere-p, (f) basal part of phallus – bp, a – aedeagus, c – cauda.

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as body sclerotization in all known morphs. The main general character distinguishing the European species is the presence or absence of the dorsal scleroites at setae bases, which distinguishes E. cembrae as the least typical species due to the unclerotised abdomen and a few inconspicuous setae on the dorsal side of body – “cembrae” group. Among all Eulachnus species, only two other taxa from Japan and China (E. pumilae Inouye, 1939 and E. piniarmandifoliae Zhang, 1992) are similar to E. cembrae due to the absence of the dorsal scleroites. This extraordinary character has been a reason for confusion over the identity of E. cembrae (Remaudière and Remaudière, 1997; Mamontova, 2012; Kanturski and Wieczorek, 2014b) for years. E. cembrae, like other European species from this genus, are characterized by the absence of accessory setae on the apical segment of the rostrum, while some Asiatic species like E. pumilae or E. thunbergi (Wilson, 1919) have one pair of accessory setae on the ARS. Due to the absence of dorsal sclerotization, E. cembrae and other species from this group are the best recognizable species in this genus. Other representatives of the European species of Eulachnus are characterized by the presence of scleroites at the setae bases on the dorsum, but ﬁrst of all they can be distinguished by differently developed setae on head, antennae, dorsum and legs. The most “hairy” species group, with ﬁne or often rigid setae, belong to the “agilis” group. Morphological analysis using the light

microscope conﬁrmed the hypothesis formulated by Binazzi (1983) and Mier-Durante and Binazzi (1997) that the “agilis” group includes the most similar species: E. agilis, E. rileyi, E. alticola Börner, 1940, E. tuberculostemmatus, E. mediterraneus and E. intermedius (Kanturski et al., in preparation). The species from this group examined in this study (E. agilis, E. rileyi, E. tuberculostemmatus) present the most extreme characters in the type of chaetotaxy: E. rileyi with the longest, ﬁne, hair-like setae; E. agilis with long, rigid and pointed or slightly blunt setae and E. tuberculostemmatus with the shortest dorsal and slightly capitate setae on antennae and legs. Species in this group have a tendency to shortening of dorsal setae, from the longest in E. rileyi and E. agilis to the shortest presented in E. tuberculostemmatus, in which the dorsal setae are nearly as short as those in the species of the “brevipilosus” group. However, long, rigid and slightly pointed setae on tibiae and head clearly place this species in the “agilis” group, but as the least typical taxon (Table 2). On the contrary, two species of the European Eulachnus, due to very short, often thick, rigid and capitate setae, form the third morphological species group – the “brevipilosus” group. Species from this group are the smallest aphids in this genus, they are almost “naked” and often have shorter tibiae. Field observations have conﬁrmed that E. brevipilosus and E. nigricola, unlike other European Eulachnus species which are living on the upper side of the needle, feed behind the needles near the basal part of the cluster. This leads

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Fig. 12. SEM of genitalia of the male of E. nigricola. (a) head of the basal part of phallus, (b) rough surface of the basal part of phallus, (c) structures on the surface of the basal part of phallus, (d) location of sensilla at the base of paramere, (e) different shape of heads of the sensilla, (f) structure of the sensilla (HMDS). 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437

to a situation where the feeding aphid sits very close to the needles and long, rigid setae could disturb the aphid in the occupation of the most suitable place on the host plant. This hypothesis is supported by the fact that both E. brevipilosus and E. agilis are living on the same host plants – Pinus silvestris or P. mugo and also both E. nigricola and E. rileyi are associated with P. nigra. Therefore, these aphid species occupy separate ecological niches. This is also supported by the ecological speciation rule known in the tribe Eulachnini and the family Lachninae (Favret and Voegtlin, 2004; Jousselin et al., 2013; Depa et al., 2014, in press). The alate viviparous females of the European species of the genus Eulachnus are recognized as rare, occurring at the end of spring and in early summer in small numbers, for example, the alate of E. cembrae have been recorded only once in high Tatra Mountains by Paˇsek (1954). Because of their rarity, we could compare only the alate morphs from the “agilis” and “brevipilosus” groups. The winged females demonstrate the most characteristic features for species and species groups, which are manifested by similar differences in chaetotaxy and sclerotization. Also Paˇsek (1954) reported that the alate of E. cembrae are characterized by the lack of dorsal scleroites on a “raw” abdomen. Moreover, in opposition to the European Eulachnini and other Lachninae, the alatae of Eulachnus are not much larger than the apterous females and are the smallest morphs among all Lachnids. The representatives of the sexual generation (oviparous females and males) are the most rare morphs,

occurring only once a year for a short time in autumn and in the temperate climate (Wieczorek et al., 2013). As the offspring of the parthenogenetic females, they do not differ from apterous and alate females in general morphology and present the main morphological characters signiﬁcant for each species group. 4.2. Morphology focus The sensory system of aphids are the least known in all Hemiptera. Until recent times, only the sensilla of Heteroptera and ˙ and Chłond, Fulgoromorpha have been the best understood (Brozek ˙ 2010; Brozek and Bourgoin, 2012; Dai et al., 2014). Very little is known about the sensory organs of aphids other than the antennal rhinaria (Bromley et al., 1979, 1980; Sun et al., 2013) and prioprioreceptors (Pringle, 1938). The most interesting sensilla found on the area between trochanter and femur in alate viviparous females of E. brevipilosus can be similar to peg sensilla without pores as in Sogatella furcifera (Delphacidae) (Dai et al., 2014) or nonporous ﬁnger-like sensilla as in Encarisia guadeloupae (Aphelinidae) (Zhou et al., 2013). These can be most likely treated as mechanoreceptors, the more so as they are extremely exposed on the pillow-like protuberance on the membranous part of the articulation. The sensilla found in a similar area in E. rileyi, which are only about three, are lying in a cavity, resembling dome-shaped sensilla without pores ˙ as in semiaquatic bugs (Gerromorpha) (Brozek and Zettel, 2014),

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Fig. 14. SEM of ventral end of abdomen of oviparous female of E. rileyi. (gp) genital plate, ap-anal plate, (e) egg locked in the genital aperture, (c) cauda (HMDS).

4.3. Methodology

Fig. 13. SEM of surface of hind tibiae of oviparous females of the genus Eulachnus. (a) E. rileyi (CPD), (b) E. brevipilosus (CRYO), (c) pseudosensorium of E. nigricola (HMDS); ps – pseudosensoria.

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are completely different, so it is probable that they can be higroreceptors. As this new sensory structure occurs in the two lineages related of the same genus (i.e. only in winged forms of E. brevipilosus and E. rileyi) and in the same place though looking different, is however most probably hom*ologous, despite the fact that single sensilla are morphologically different and in various number. This structure has not been observed in apterous females (viviparous and oviparous) or males. There is also no information about sensory structures on the genitalia of aphids, so it is very difﬁcult to compare the sensilla on the basal part of the phallus of E. nigricola with any other known structures in other groups of Hemiptera. It is possible that the structures may play a role during the copulation process, as discovered in differently-looking sensilla in Coleoptera male genitalia (Düngelhoef and Schmitt, 2010). Undoubtedly, further and detailed studies should be conducted to obtain more data about these structures, hitherto unknown in this group of insects.

In scanning electron microscopy methodology, critical point drying with chemical ﬁxation in glutaraldehyde (CPD) is the most common method of sample drying and it is considered by many to be the best method to avoid surface tension artifacts in dried specimens (Swearingen et al., 1997; Pathan et al., 2008; Talbot and White, 2013). An alternative to CPD is hexamethyldisilazane (HMDS), which is a rapid, inexpensive method for the preparation of soft biological samples for SEM. Some authors claimed that HMDS treatment did not cause disruption of cell structure and was therefore suitable for drying delicate and wet samples (Braet et al., 1997; Araujo et al., 2003; Jung et al., 2010). Despite these potential advantages, CPD is still the most frequently used method of drying soft and hydrated specimens for SEM. Unlike the CPD and HMDS methods, cryo-SEM allows for imaging of samples in a frozen hydrated state, and thus eliminates ﬁxation and drying artifacts (Kaminskyj and Dahms, 2008). In our study, HMDS without glutaraldehyde ﬁxation and cryoSEM preparation methods provided acceptable image quality and minimal artifacts compared with the CPD technique. For the examined adult specimens, HMDS and cryo-SEM images showed intact surface morphology and therefore these preparation methods are suitable for delicate tissues of aphids. In the HMDS drying process, the specimens were immersed in HMDS for 30 min; therefore, the time of drying and then leaving the specimens in the desiccator to air-dry at room temperature is important in the removal of artifacts. This is consistent with other studies that have shown the efﬁcacy of HMDS (Lee and Chow, 2012). Some authors claimed that HMDS treatment did not cause disruption of surface structure and was therefore suitable for drying soft biological samples (Kashi et al., 2014). Cryo-SEM was also found to be useful for imaging surface morphology in aphid samples given the resolution requirements, the natural presence of epicuticular waxes and the ﬁne surface structures that are extremely susceptible to thermal damage. This technique can be used to image biological samples in a frozen hydrated state, and thus eliminates ﬁxation and drying artifacts (Craig and Beaton, 1996; McCully et al., 2009; Garzo et al., 2012). Although cryo-SEM technique can potentially produce images at high magniﬁcation and without dehydration artifacts, it is also prone to incomplete sublimation of surface moisture, which

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In our study, the differences in surface tension of the prepared and preserved samples are most probably due to the time of collection as well as the kind of morph. Apterous viviparous females collected in the spring were better ﬁlled by numerous embryos, because individuals of most species of Eulachnus have two periods of abundance: in the spring, when the development is intensive, and in the autumn when the oviparous females are ﬁlled with fertilized eggs. We also observed that the maintenance of surface tension of head and thorax structures of the alate viviparous females and alate males was a result of stronger sclerotization of those structures. Therefore, it seems that a properly selected method combined with the knowledge of the biology of the studied species, can be the best approach to preservation of highly hydrated and delicate small arthropods. Acknowledgements We are grateful to Justyna Płoszaj-Pyrek (Faculty for Biology and Environmental Protection, University of Silesia) for technical assistance in the cryo-SEM analysis. Special thanks go to Marcin Walczak and Artur Taszakowski (Department of Zoology, University of Silesia) for collecting the E. tuberculostemmatus samples and Paul A. Brown (Natural History Museum, London) for linguistic assistance. We would like to express our thanks to the Editor and the Reviewers for all valuable suggestions and comments that have improved the manuscript. References

Fig. 15. High magniﬁcation of SEM of the surface of antennal segment III of E. brevipilosus: (a) CRYO, (b) HMDS, (c) CPD.

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obscures ﬁne surface features (e.g. Jansson et al., 2000; Kaminskyj and Dahms, 2008). In addition, this approach requires highly specialized equipment and lengthy sample preparation (BouchetMarquis and Fakan, 2009). Larger magniﬁcation (22 k) have shown that the compared methods do not affect the visible differences in the preservation of the surface (Fig. 15) that can be important in the choosing of different method to the best preservation of biological samples for morphological study.

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Morphology of the European species of the aphid genus Eulachnus (Hemiptera: Aphididae: Lachninae) - A SEM comparative and integrative study. - PDF Download Free (2024)