Вы находитесь на странице: 1из 43

NEMATODE WORKSHOP:

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

NEMATODE WORKSHOP:
Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control
June 12-13, 2005 Locorotondo, Bari, Italy,

Sponsored by
10th European Meeting of the IOBC/WPRS Working Group "Insect Pathogens and Insect Parasitic Nematodes" in cooperation with COST ACTIONS 842 and 850 Entomophthorales & Biocontrol Symbiosis

WORKSHOP MANUAL
Prepared by:
Dr. S. Patricia Stock
Division of Plant Pathology and Microbiology Department of Plant Sciences Department of Entomology University of Arizona, Tucson, AZ, USA

This manual is to be used only for scientific teaching purposes. It does not constitute a scientific publication, and may not be used for any commercial activity. Unauthorized reproduction may violate copyrights held by the author or by the copyright holders of the illustrations reproduced in this manual.

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

TABLE OF CONTENTS

A. Classification B. Diagnosis of major groups C. Tables D. Polytomous keys E. Preparation of Nematodes for Identification F. Atlas of main morphology and terminology G. Molecular Diagnostics / Phylogenetics H. References

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

A. Classification _____________________________________________________________
More than 30 nematode families are known to host taxa that parasitize or are associated with insects (Maggenti, 1981; Nickle, 1972; Poinar, 1975, 1983, 1990; Kaya and Stock, 1997). However, because of their biological control potential, research has concentrated on seven families: Mermithidae, Allantonematidae, Neotylenchidae, Sphaerularidae, Rhabditidae, Steinernematidae and Heterorhabditidae, the latter two families currently receiving the most attention as control agents of soil insect pests (Lacey et al., 2001). The biological control potential of nematodes is not restricted to insects. Phasmarhabditis hermaphrodita (Schneider), a member of the family Rhabditidae, is known to suppress several slug species, and has recently been developed as a biological molluscicide. Several predatory nematodes such as mononchids, dorylaimids, nygolaimids, diplogasterids have also potential for management of plant-parasitic nematodes. Moreover, the fungal-feeding aphelenchid, Aphelenchus avenae Bastian, has also been considered as potential biological control agent of soilborne fungal pathogens of plants. I have adopted the new classification scheme suggested by De Ley and Blaxter (2002) to list those groups with biological control potential. This classification is rooted on a phylogenetic interpretation of a preliminary evolutionary tree based on 18S ribosomal DNA proposed by Blaxter et al. (1998). This molecular framework recognizes the presence of three basal clades: 1) dorylaimids,2) enoplids and 3) chromadorids. Relationships between these clades have not been fully resolved yet, but available data support sister taxon status of dorylaims and enoplids (De Ley and Blaxter, 2002). In this new taxonomic scheme, dorylaims and enoplids are grouped within the class Enoplea Inglis, 1983. The Chromadorea Inglis, 1983 comprise the majority of taxa within Nematoda, including all the former Secernentea. In this classification system, seven out of eleven nematode families currently considered in biological control are grouped within the Chromadorea, the remaining families, the Mononchidae, Mermithidae, Dorylaimidae and Nygolaimidae are members of the Enoplea (Table 1).

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

B. Diagnosis of major groups


____________________________________________________________________________________ CLASS CHROMADOREA Family Steinernematidae Chitwood and Chitwood, 1937 Diagnostics: Adults with truncated to slightly round head. Lips fused six, but tips distinct, and each with one labial papilla. Four cephalic papillae present. Amphids small. Stoma reduced, short and wide, with inconspicuous sclerotised walls. Oesophagus rhabditioid, set off from intestine. Nerve-ring usually surrounding isthmus or anterior part of basal bulb. Excretory pore opening distinct. Female with paired opposed ovaries. Vagina short, muscular. Vulva located near middle of body, with or without protruding lips. Epiptygma present or absent. Male with single reflexed testis. Spicules paired, symmetrical. Gubernaculum present. One single midventral and 10-14 pairs of genital papillae present of which 7-10 pairs precloacal. Tail rounded, digitated or mucronated. Third-stage infective juvenile with collapsed stoma. Cuticle annulated, lateral field with 6-8 ridges in middle of body. Oesophagus and intestine collapsed. Specialized bacterial pouch located at beginning of intestine and of variable shape. Excretory pore distinct, anterior to nerve ring. Tail conoid or filiform, with variable hyaline portion. Phasmids present, prominent or inconspicuous. The Steinernematidae currently comprise 2 genera, Steinernema Travassos, 1927 with more than 30 species and Neosteinernema Nguyen and Smart, 1994 with only one species N. longicurvicauda (Table 2) Bionomic Notes: Steinernematids are obligate pathogens in nature and are characterized by their mutualistic association with bacteria of the genus Xenorhabdus. Of all nematodes studied for biological control of insects, the Steinernematidae together with the Heterorhabditidae, have received the most attention because they possess many of the attributes of effective biological control agents.

Figure 1. Steinernema monticolum (From Stock et al, 1998). A. Male in toto, B. Male tail, C. Female, stomatal region, D. Female in toto, E. Infective juvenile, anterior end, F. Infective juvenile, tail.

Family Aphelenchidae Fuchs, 1937 Diagnostics: Labial cap distinct and often set off by a constriction. Hollow axial protrusible spear with slight basal thickenings. Oesophagus with a large metacorpus (median bulb). Dorsal

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

oesophageal gland opening into metacorpus. Oesophageal glands either forming a lobe or adjoined intestine. Male bursa supported by four pairs of caudal papillae (rays). Spicules ventrally arcuate and slender. Gubernaculum present.

Diagnostics: Preparasitic female and free-living male with small stylet (less than 15 m long) with or without knobs. Oesophageal glands elongated, lobe-like; subventral glands extending past dorsal lobe. Tail conoid or subcylindrical. Preparasitic female with small vulva and short vagina. Postvulval sac short or absent. Uterus elongated. Parasitic female obese, sac-like, elongate or spindleshaped. Reproductive organs filling body cavity. Uterus not everted. Vulva a small transverse slit or indistinct. Male with outstretched testis. Spicules arcuate, pointed, usually less than 25 m long. Gubernaculum usually present. Bursa present or absent.

B
Figure 3. Thripinema rineraoi. A, F: Anterior and posterior region of partially free-living impregnated female; B: Male; C, D: Anterior and posterior regions of male; E: Entomoparasitic female from haemocoel of Megaluriothrips sp. (After Siddiqi, 1986, courtesy CAB International).

Figure 2. Aphelenchus avenae Bastian. (After Stock and Hunt, 2005). A: Female; B: Male tail, lateral view.

Bionomic Notes: Aphelenchids are fungal-feeding nematodes. They can be found in decaying plant tissues feeding on various fungal hyphae. Until now, Aphelenchus avenae has been studied as a biological control alternative to suppress fungal pathogens of plants. Family Allantonematidae Pereira 1931

Bionomic Notes: Allantonematids have a single heterorsexual life cycle. Females are obligate parasites of the haemocoel of mites and insects. Within this family, members of Thripinema Siddiqi, 1986 are known to parasitize thrips (Thysanoptera: Thripidae). A free-living stage occurs in flowers, buds and leaf galls of plants that attack thrips.

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

Family Neotylenchidae Thorne, 1941 Diagnostics: (modified from Siddiqi, 2000). Free-living stages with smooth or finely striated cuticle. Stylet well developed, less than 20 m long, basal knobs may be bifid. Oesophagus fusiform, basal bulb absent. Oesophageal glands free in body cavity, extending over intestine. Orifice of dorsal gland close to stylet base. Nerve ring generally circumintestinal, posterior to, or at level of, oesophago-intestinal junction. Excretory pore anterior or posterior to nerve ring. Female monodelphic prodelphic. Vulva in posterior region, postvulval sac present or absent. Tail conoid, subcylindroid or cylindroid. Male monorchic, testis outstretched. Bursa present or absent. Spicules paired, small, cephalated or arcuate, distally pointed. Gubernaculum present or absent. Preadult female (free-living) with hypertrophied stylet and oesophagus. Ovary immature. Uterus long. Mature parasitic female, obese, sausage-shaped or elongate tuboid. Stylet and oesophagus non-functional. Uterus hypertrophied but not everted.

Bionomic Notes: Neotylenchids are characterized by having a free-living generation alternating with an insect parasitic generation. Beddingia Thorne, 1941 currently comprises 17 nominal species with B. siricidicola Bedding, 1968, a parasite of the wood wasp Sirex noctilio, being the only taxon currently used in biological control

Family Rhabditidae rley, 1880 Diagnostic Characters: Stoma commonly cylindrical without distinct separation of cheilo-, gymno- and stegostom. Stoma two or more times as long as wide. Usually with six distinct lips, each with one cephalic papilla. Amphids porelike. Oesophagus clearly divided into corpus (procorpus and metacorpus) and postcorpus (isthmus and valvated muscular portion). Male spicules separate or fused distally. Gubernaculum present. Bursa mostly well developed, peloderan or leptoderan, occasionally small or rudimentary. Nine or ten pairs of genital papillae (bursal rays). Female with one or two ovaries.

Figure 4. Beddingia (=Deladenus) siricidicola Bedding (From Stock and Hunt, 2005). A: Oesophageal region of fungus feeding female; B: Oesophageal region of entomoparasitic preadult female; C: Male tail region; D: Posterior region of fungus feeding female. (After Siddiqi, 2000, courtesy CAB International).

Figure 5. Phasmarhabditis hermaphrodita (Schneider). A-B. Female, stoma dorsal view (A) and subventral (B); C. Oesophagus; D-E. Female tail, lateral view (D) and ventral (E); F. dauer juvenile,

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005. anterior regionl, G. Female gonad, anterior end, L. Male tail, lateral view. (From Hooper et al., 1999)

Bionomic Notes: Most members of this family are free-living bacterivores, but, two species of Phasmarhabditis, P. hermaphrodita (Schneider, 1859) and P. neopapillosa (Schneider, 1866) have parasitic associations with terrestrial slugs and snails. Phasmarhabditis hermaphrodita is capable of killing several slug and snail pests and is the only species currently used as a biological control agent and is mass produced and commercialized as a molluscicide (See Wilson and Grewal, Chapter 23 and Glenn and Ester, Chapter 24). Family Heterorhabditidae Poinar, 1976 Diagnostics: Adults with six distinct protruding pointed lips surrounding oral aperture. Each lip bearing one labial papilla. Stoma short and wide. Oesophagus rhabditoid. Corpus cylindrical, metacorpus not differentiated. Isthmus short. Basal bulb pyriform with reduced valve. Excretory pore usually located at level of basal bulb. Hermaphrodite (first generation) with an ovotestis. Vulva located near midbody. Postanal swelling present or absent. Tail terminus blunt, with or without a mucro. Female (second generation) amphidelphic, ovaries with reflexed portions often extending past vulva opening. Vulva located near middle of body, with or without protruding lips. Tail conoid; postanal swelling present or absent. Male (second generation) monorchic. Spicules paired, symmetrical, straight or arcuate, with pointed tips. Gubernaculum slender, about half length of spicules. Bursa open, peloderan, attended by a complement of nine pairs of bursal rays (papillae). Thirdstage infective juvenile ensheathed in cuticle of second-stage juvenile. Cuticle of J2 with longitudinal ridges throughout most of body length, and a tessellate pattern in anterior-most region. Lateral field with two ridges. Prominent cuticular dorsal tooth present. Excretory pore

located posterior to basal bulb. Tail short, conoid, tapering to a small spike-like tip. Heterorhabditidae consists of one genus, Heterorhabditis Poinar, 1976, with H. bacteriophora as the type and 10 other species described (Table 3).

Figure 6. Heterorhabditis downesi Stock et al. (from Stock et al. 2003) . A. Hermaphrodite; B. Spicules; C. Gubernaculum; D. Hermaphrodite,vuvla; E. Male tail, ventral view; F. Male, anterior end; G. Female tail.

Bionomic Notes: Heterorhabditids have a similar life cycle to steinernematids, but adults resulting from infective juveniles are hermaphroditic. Eggs laid by the hermaphrodites produce juveniles that develop into males and females or infective juveniles. The males and females mate and produce eggs that develop to infective juveniles.

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

Family Diplogasteridae Micoletzky, 1922 Diagnostics: Lip region, never set off by a constriction, usually composed of six distinct lips or six fused lips. Amphids pore-like. Stylet absent. Stoma variable, usually broad and short with stegostom containing denticles, warts or teeth. Oesophagus with a median valvated bulb and a basal valveless bulb. Female gonad usually paired. Male with paired spicules and gubernaculum. Bursa usually small or absent. Male tail often with nine pairs of genital papillae and a pair of phasmids. Three pairs of genital papillae located preanal.

CLASS ENOPLEA Family Mononchidae Chitwood, 1937 Diagnostics: Generally large, stout nematodes. Cuticle usually appearing nonstriated and smooth. Lateral field usually not differentiated. Head not distinctly offset, composed of 6 or fewer confluent lips, each carrying at least two papillae. Amphids small, cup-shaped. Stylet absent. Stoma forming a small to large barrelshaped cuticularized chamber bearing an immovable dorsal tooth. Subventral teeth and/or rows of denticles or ridges may also be present. Oesophagus stout, muscular, glandular and almost cylindrical with some posterior swelling. Oesophago-intestinal junction tuberculate or non-tuberculate. Excretory pore usually absent. Females usually with paired ovaries, opposite and reflexed. Males with paired opposed testes leading to a common vas deferens. Spicules paired. Gubernaculum present. Lateral guiding piece often present. Midventral row of precloacal papillae always present on males. Tail variable in form. Bursa absent A C

Figure 7. Aduncospiculum halicti Giblin y Kaya A. Adult, anterior end; B-C. Male tail, ventral view (B), and lateral (C); Female tail, lateral view; E. Female, middle-body region. (From Giblin and Kaya)

B D

Bionomic Notes: Most Diplogasteridae members are predators or omnivores but a few taxa are also bacterial feeders. Of all of them, only a few genera (i.e. Butlerius, Fictor and Mononchoides) have been studied as biological control agents of plant parasitic nematodes

Figure 8. Mononchidae. A-B Mononchus sp. A. anterior region; B: female tail region. C-D Mylonchulus minor. C. Anterior region; D: Female tail region. Modified from Stock and Hunt, 2005.

Bionomic Notes: Mononchids are predominantly predaceous nematodes feeding on small invertebrates (including other nematodes) in soil and fresh water. Many genera have been proposed, but only Mylonchulus (Cobb, 1916), Mononchus

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

Bastian and Iotonchus (Cobb, 1916) have been explored as biocontol agents. A few taxa have been used against plantparasitic nematode species Family Mermithidae Braun, 1883 Diagnostics: Long slender nematodes sometimes reaching a length of 50 cm, but usually between 1 and 10 cm. Cuticle smooth or with criss-cross fibers. Anterior end containing two, four or six cephalic papillae and rarely a pair of lateral mouth papillae. Amphids tube-like or modified pouch like. Oesophagus modified into a slender tube surrounded posteriorly by stichosomal tissue. Intestine modified into a trophosome or food storage organ forming a blind sac soon after the nematodes enter a host. Preparasitic juveniles (J2) with a functional stylet and a pair of penetration glands that degenerate after host invasion. Ovaries paired; muscular vagina straight or curved. Males with a single fused or paired spicules. Gubernaculum and bursa absent. Several rows of genital papillae usually present

constructed. All known species are obligate parasites of terrestrial and aquatic arthropods and other invertebrates. Mermithids parasitize many different insect groups, including Orthoptera, Dermaptera, Hemiptera, Lepidoptera, Diptera, Coleoptera, and Hymenoptera. Mermithids with significant biological control potential include Romanomermis culicivorax, a parasite of mosquito larvae (Petersen, 1984, 1985), Oesophagomermis (=Filipjevimermis) leipsandra, a parasite of larval banded cucumber beetle Diabrotica balteata (Creighton and Fassuliotis, 1983), Mermis nigrescens, a parasite of grasshoppers (Webster and Thong, 1984) and Agamermis unka, a parasite of white and brown planthoppers (Choo et al., 1989, 1994)

Figure 10. Romamnomermis culicvorax. A. Female head, B. postparasitic juvenile tail, C. Male papillae pattern, D. Female vagina, E. egg, F. Male tail, G. female tail, H. preparasitic larva, I. Postparasitic juvenile stoma showing tooth, J. parasitized mosquito. (From Nickle, 1972). Figure 9. Mermithidae 1.Mermis nigrescens Dujardin. A. lateral view of female head, B. Postparasitic juvenile tail, C. Preparasitic larva, D. Vagina, lateral view, E. egg with byssi, F. Male tail, lateral view, G. female tail, lateral view, H. Parasitized grasshopper (From Nickle, 1972).

Bionomic Notes: There are numerous described genera, many of which are poorly characterised by contemporary standards. The group is in urgent need of revision before a workable key can be

10

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

Family Dorylaimidae De Man, 1876 Diagnostics: Generally large and robust nematodes. Stoma with an axial odontstyle, the aperture of which is located dorsally. Oesophagus cylindrical and divided into two parts: anterior portion usually slender, sometimes with small muscular swellings, followed by an expanded posterior portion. Excretory pore rudimentary or absent. Females with one or two ovaries. Males with two testes. Spicules robust and separated. Gubernaculum usually absent, but lateral guiding pieces present. Bursa absent. Setae and caudal glands absent.

Family Nygolaimidae Thorne, 1935 Diagnostics: Stoma armed with mural tooth of variable shape. Dorylaimoid oesophagus with posterior portion enclosed in a sheath. Three large cardiac glands at oesophag-intestinal junction. Ovaries paired, opposed and reflexed. Spicules arcuate. Gubernaculum and lateral guiding pieces present in some males. Bionomic Notes: Likewise dorylaimids, nygolaimids are predaceous, a few taxa considered for biological control of plantparasitic nematodes

Figure 11. Dorylaimidae. A-C, Mesodorylaimus. A: Head region; B: Female tail; C: Male tail. D-F, Allodorylaimus. D: Head region; E: Female tail; F: Male tail. G-J, Eudorylaimus. G: Head region; H: Vulval region; I: Male tail, J: Female tail. K, L, Discolaimus. K: Pharyngeal region; L: Head region. M-O, Labronema. M: Head region; N. Male tail region; O: Female tail. P-R, Pungentus. P: Head region; Q: Female tail; R: Male tail region. (After Jairajpuri & Ahmad, Dorylaimida. Free-living, Predaceous and Plant-parasitic Nematodes (1992)

Bionomic Notes: The feeding habits of many members are not known, although some are acknowledged as being predaceous on other nematodes and invertebrates.

11

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

C. TABLES
____________________________________

12

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

Table 1. Major groups in Nematoda with biological control potential. (Classification based on De Ley and Blaxter, 2002)
CLASS SUBCLASS ORDER SUBORDER Mononchida Mononchina Jairajpuri, 1969 Kirjanova y Krall, 1969 Dorylaimida Nygolaimia Pearse, 1942 Thorne, 1935 Mermithida Mermithina Hyman, 1951 Andrssy, 1974 INFRAORDER SUPERFAMILY Mononchoidea Chitwood, 1937 Nygolaimoidea Thorne, 1935 Mermithoidea Braun, 1883 FAMILY Mononchidae Chitwood, 1937 Nygolaimidae Thorne, 1935 Mermithidae Braun, 1883

ENOPLEA Inglis 1983

DORYLAIMIA Inglis, 1983

Panagrolaimomorpha De Ley y Blaxter, 2002 Tylenchina Thorne, 1949 CHROMADOREA Inglis, 1983 CHROMADORIA Pearse, 1942 RHABDITIDA Chitwood, 1933 Tylenchomorpha De Ley y Blaxter, 2002

Strongyloidoidea Chitwood y McIntosh, 1934 Aphelenchoidea Fuchs, 1937 Sphaerularoidea Lubbock, 1861

Steinernematidae Travassos, 1927 Aphelenchidae Fuchs, 1937 Allantonematidae Pereira, 1931 Neotylenchidae Thorne, 1941 Rhabditidae rley, 1880 Heterorhabditidae Poinar, 1975 Diplogasteridae Micoletzky, 1922

Rhabditina Chitwood, 1933

Rhabditomorpha De Ley y Blaxter, 2002 Diplogasteromorpha De Ley y Blaxter, 2002

Rhabditoidea rley, 1880 Strongyloidea Baird, 1853 Diplogasteroidea Micoletzky, 1922

* Families within Sphaerularoidea are listed based on the classification proposed by Siddiqi (2000) which recognizes three families within the Sphaerularoidea: Sphaerulariidae, Lubbock, 1861; Allantonematidae, Pereira, 1931 and Neotylenchidae Thorne, 1941

13

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

Table 2. Taxonomic summary of the family Steinernematidae (Modified from Stock and Hunt, 2005). Family Steinernematidae Chitwood and Chitwood, 1937 Syn. Neoaplectanidae Sobolev, 1953 Taxa Type genus: Steinernema Travassos, 1927 Type species: Steinernema kraussei (Steiner, 1923) Travassos, 1927 Other species: S. abbasi Elawad, Ahmad and Reid, 1997 S. affine (Bovien, 1937) Wouts, Mrcek, Gerdin and Bedding, 1982 S. anatoliense Hazir, Stock and Keskin, 2003 S. apuliae Triggiani, Mracek and Reid, 2004 S. arenarium (Artyukhovsky, 1967) Wouts, Mrcek, Gerdin and Bedding, 1982 S. asiaticum Anis, Shahina, Reid and Rowe, 2002 S. bicornutum Tallosi, Peters and Ehlers, 1995 S. carpocapsae (Weiser, 1955) Wouts, Mrcek, Gerdin and Bedding, 1982 S. caudatum Xu, Wang and Li, 1991 S. ceratophorum Jian, Reid and Hunt, 1997 S. cubanum Mrcek, Hernandez and Boemare, 1994 S. diaprepesi Nguyen and Duncan, 2002 S. feltiae (Filipjev, 1934) Wouts, Mrcek, Gerdin and Bedding, 1982 S. glaseri (Steiner, 1929) Wouts, Mrcek, Gerdin and Bedding, 1982 S. hermaphroditum Stock, Griffin and Chaenari, 2004 S. intermedium (Poinar, 1985) Mamiya, 1988 S. karii Waturu, Hunt and Reid, 1997 S. kushidai Mamiya, 1988 S. loci Phan, Nguyen and Moens, 2001 S. longicaudum Shen and Wang, 1992 S. monticolum Stock, Choo and Kaya, 1997 S. neocurtillae Nguyen and Smart, 1992 S. oregonense Liu and Berry, 1996 S. pakistanense Shahina, Anis, Reid, Rowe and Maqbool, 2001

Geographic Origin* Europe (Germany), North America Asia (Oman) Europe (Denmark) Asia (Turkey) Europe (Italy) Asia (Central Russia) Asia (Pakistan) Europe (Yugoslavia) Asia, Europe (Czechoslovakia), North America, South America Asia (China) Asia (China) Central America (Cuba) North America (USA) Europe (Denmark), North America, South America Asia, Europe, North America (USA), South America Asia (Indonesia) North America (USA), Europe Africa (Kenya) Asia (Japan) Asia (Vietnam) Asia (China), North America Asia (Korea) North America (USA) North America (USA) Asia (Pakistan)

14

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

S. puertoricense Romn and Figueroa, 1994 S. rarum (de Doucet, 1986) Mamiya, 1988 S. riobrave Cabanillas, Poinar and Raulston, 1994 S. ritteri de Doucet and Doucet, 1990 S. sangi Phan, Nguyen and Moens, 2001 S. scapterisci Nguyen and Smart, 1990 S. scarabaei Stock and Koppenhfer, 2003 S. serratum Liu, 1992 S. siamkayai Stock, Somsook and Kaya, 1998 S. tami Van Luc, Nguyen, Spiridonov and Reid, 2000 S. thanhi Phan, Nguyen and Moens, 2001 S. thermophilum Ganguly and Singh, 2000 S. websteri Cutler and Stock, 2003 S. weiseri Mrcek, Sturhan and Reid, 2003 Genus: Neosteinernema Nguyen and Smart, 1994 Type and only species: Neosteinernema longicurvicauda Nguyen and Smart, 1994 * Country of original isolation in parenthesis species inquirenda

Central America (Puerto Rico) South America (Argentina), North America North America (USA) South America (Argentina) Asia (Vietnam) South America (Uruguay) North America (USA) Asia (China) Asia (Thailand) Asia (Vietnam) Asia (Vietnam) Asia (India) Asia (China) Europe (Germany) North America (USA)

15

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

Table 3. Taxonomic summary of described Heterorhabditis species (Modified from Stock and Hunt, 2005).
Taxa Type and only genus: Heterorhabditis Poinar, 1976 Syn. Chromonema Khan, Brooks and HIrschmann, 1976 Type species: Heterorhabditis bacteriophora Poinar, 1976 Syn. Chromonema heliothidis Khan, Brooks and Hirschmann, 1976 H. heliothidis (Khan, Brooks and Hirschmann, 1976) Poinar, Thomas and Hess, 1977 H. argentinensis Stock, 1993 Other species: H. baujardi Phan, Subbotin, Nguyen and Moens, 2003 H. brevicaudis Liu, 1994 H. downesi Stock, Burnell and Griffin, 2002 H. indica Poinar, Karunakar and David, 1992 Syn. H. hawaiiensis Gardner, Stock and Kaya, 1994 H. marelata Liu and Berry, 1996 Syn. H. hepialius Stock, Strong and Gardner, 1996 H. megidis Poinar, Jackson and Klein, 1987 H. mexicana Nguyen , Shapiro-Ilan, Stuart, McCoy, James and Adams, 2004 H. poinari Kakulia and Mikaia, 1997** H. taysearae Shamseldean, Abou El-Sooud, Abd-Elgawad and Saleh, 1996 H. zealandica Poinar, 1990 Asia (Vietnam) Asia (China) Europe (Ireland) Asia (India), Central America, North America North America (USA) North America (USA), Europe North America (Mexico) Europe (Georgia) Asia (Egypt) Australia (New Zealand) Africa, Asia, Australia, Central America, Europe, North America (USA) , South America Biogeography*

* Country of original isolation in parenthesis; ** Species inquirenda. As proposed by Stock (in press).

16

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

D. Polytomous keys

17

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

Key 1. Polytomous key for Steinernematidae. All data from original descriptions unless otherwise specified. Morphometrics are given in microns (From Stock and Hunt, 2005)
Neosteinernema Key Diagnostic Features: Adults and third-stage infective juvenile with very conspicuous amphids. Male with ventrally arcuate spicules with a very prominent manubrium. Third-stage juvenile with very long (as long as oesophagus length) and filiform tail. Third Stage Infective Juvenile First Generation Adults Male Species longicurvicauda L 920 789-1084 W 24 20-31 EP 68 61-76 T 167 141-190 D% 41 38-46 Female E% LF SPL GL SW D% M EPI VL 41 8 61 59 1.03 NA A A V 37-48 52-67 52-66 0.8-1.15 Steinernema Key Diagnostic Features: Adults and third-stage infective juvenile with phasmids not visible. Shape of spicules variable but not with a manubrium shape as in Neostreinernema. Third-stage infective juvenile with conoid tail variable in size. Third Stage Infective Juvenile First Generation Adults Male Species asiaticum siamkayai ritteri rarum tami abbasi anatoliense thermophilum TBL 425* 360-450 446 398-495 510 470-590 511 443-573 530 400-600 541 496-579 545 507-580 555 510-620 MBW 23 20-25 21 18-24 22 19-24 23 18-26 23 19-29 29 27-30 24.5 21-28 21 21-23 EP 32 28-34 35 29-38 43 40-46 38 32-40 36 34-41 48 46-51 37 36-39 40 37-46 TL NA 36 31-41 49 44-54 51 4-56 50 42-57 56 52-61 52 46-58 45 40-52 E% LF SPL carpocapsae-group (IJ average size < 600 m) 32* 78* 6 68* 30-36 60-90 61-74 37 96 6-8 77.5 31-43 95-112 75-80 46 88 6 69 44-50 79-97 8-75 35 72 6 47 30-39 63-80 42-52 31 73 6-8 77 28-34 67-86 71-84 53 86 8 65 51-58 79-94 57-74 35 72 6 74 31.5-39 68-81.5 68-84 46 96 8 61 42-53 81-102 44-72 D% GUL 53* 46-62 54 47-65 44 33-50 34 23-38 48 38-55 45 33-50 47 42-59 36 30-42 SW * 1.7 1.4-2.2 1.56 1.44-1.57 0.94 0.91-1.05 2.0 1.4-3.0 1.56 1.07-1.87 1.75 1.6-1.9 1.7 1.2-2.8 D% P44 35-57 42 35-49 47 44-50 50 44-51 44 30-60 60 51-68 48.5 46.5-55 63 50-87 M P P A P P A P A Female EPI P P A A A P A P VL SP PR PR PR NPR PR SP PR

18

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

carpocapsae scapterisci websteri kushidai

558 438-650 572 517-609 584 553-631 589 524-662 622 561-701 671 608-800 683 649-716 693 608-880 706 591-800 706 612-821 740 586-828 753 704-784 769 648-873 849 736-950 851 720-960 885 741-988 918 890-959 932

25 20-30 24 18-30 21 17-25 26 22-31 28 26-30 29 25-32 27 24-29 30 28-34 27 23-34 37 32-46 25 24-29 35 30-40 29 25-33 26 22-29 31 27-39 34 28-42 31 25-37 33

38 30-60 39 36-38 36 29-40 46 42-50 56 51-64 65 59-69 54 49-58 62 51-69 55 47-70 58 54-62 57 43-65 51 46-54 61 53-65 62 53-67 75 68-84 18 14-22 77 72-81.5 74

53 46-61 54 48-60 47 37-56 50 44-59 54 46-59 66 53-74 58 53-62 66 64-74 66 56-74 77 71-95 60 49-68 81 76-89 72 63-78 81 70-92 63 52-72 80 64-97 76 71-80 74

riobrave intermedium pakistanense affine ceratophorum monticolum weiseri sangi bicornutum

feltiae thanhi neocurtillae scarabaei karii

60 6 66 47 54-66 58-77 39-55 73 6 83 65 60-80 72-92 59-75 77 6 68 49 62-102 64-72 42-56 92 8 63 44 84-95 NA NA intermedium-group IJ average size between 600-800 m 49 105 NA 67 51 45-55 93-111 62.5-75 47.5-56 51 96 6-8 91 64 48-58 89-108 84-100 56-75 47 91 8 68 41 42-53 87-102 62-73 36-45 49 94 8 70 46 43-53 74-108 67-86 37-56 45 84 6-8 71 40 40-56 74-96 54-90 25-45 47 76 8** 70 45 44-50 63-86 61-80 35-54 51 NA 8 68 53 44-55 62-72 46-57 40 62 8 63 40 36-44 56-70 58-80 34-46 50 84 8 62 48 40-60 80-100 53-70 38-50 feltiae-group IJ average size between 800 - 1000 m 45 78 8 70 41 42-51 69-86 65-77 34-47 58 119 8 72 49 52-67 101-138 67-78 40-56 12 23 6 58 52 10-15 18-30 52-64 44-59 60 100 8 75 44 50-75 90-110 67-83 36-50 57 96 8 83 57

26 23-28 31 27-40 31 24-34 41 38-44

1.72 1.40-2.00 2.52 2.04-2.8 1.8 1.6-2.1 1.5

41 27-55 38 32-44 40 30-50 51 NA 71 60-80 67 58-76 60 50-60 61 NA 51 33-65 55 49-61 49 39-60 49 42-63 52 50-60 60 NA 73 64-82 19 13.26 66 53-77 66

P P P A

A P A A

PR SP NP PR

1.14 1.24 NA 1.8 1.0-2.2 1.17 NA 1.4 1.0-2.0 1.4 1.2-1.5 1.8 1.5-2.4 1.5 1.2-1.6 2.22 2.18-2.26 1.13 0.99-1.3 1.8 1.5-2.1 1.43 1.18-1.64 1.7 1.5-2.0 NA

A A P P A P A P A

A A P A A A A A A

SP SP SP PR SP NP SP PR NP

P A P P A

P A P A P

PR PR V SP SP

19

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

kraussei oregonense loci

876-982 957 797-1102 980 820-1110 986 896-1072 1063 NA 1064 945-1212 1106 933-1269 1130 864-1448 1171 1057-1238 1283 1149-1508

31-35 33 30-36 34 28-38 37 30-45 40 NA 37 34-41 36 34-41 43 31-50 51 47-54 37 33-46

68-80 63 50-6 66 60-72 80 71-86 81 NA 95 86-102 82 76-89 102 87-110 95 90-102 106 101-114

64-80 79 63-86 70 64-78 75 66-83 95 NA 71 63-80 88 80-100 78 62-87 94 88-107 67 61-77

NA 47 NA 50 40-60 57 52-63

longicaudum apuliae caudatum glaseri puertoricense cubanum

NA 73-91 80 8 55 NA 52-57 100 6-8 71 90-110 65-73 107 8 71 94-120 60-80 glaseri-group IJ average size > 1000 m 56 85 8 77 NA NA NA 66 130 8 72 63-70 120-150 64-80 52 94 8 75 NA 87-100 NA 65 131 8 77 58-71 122-138 64-90 66 101 8 78 62-74 88-108 71-88 70 160 8 58 NA NA 50-67

42-64 33 23-38 56 52-59 46 40-52 48 NA 50 46-54 52 NA 55 44-59 40 36-45 39 37-42

1.10 NA 1.51 NA 1.9 1.7-2.1 1.60 1.6 1.5-1.8 2.22 2.1 1.6-2.4 1.52 1.41

57-78 53 NA 73 64-75 73 61-80 62 NA 81 75-92 71 NA 70 60-80 77 70

P A A

A A A

SP SP PR

A A A A A A

A A A A P A

PR SP-VP V PR PR PR

References: E%= EP/TL x 100; EP= excretory pore; EPI= epiptygma; D%= EP/ oesophagus length x 100; GuL= gubernaculum length; LF= number of ridges of lateral field at midbody level; M= mucro; MBW= maximum body width; SpL= spicule length; SW= SpL/ cloacal body width; TBL= total body width; TL= tail length; VL= vulval lips. A= absent; NA= not available; P= present; PR= protruding, NP= not protruding * Morphometric values of type isolate have incongruent and/or erroneous data in tables and text in original publication. **After Stock, unpublished data.

20

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

Key 2. Key diagnostic features of Phasmarhabditis spp. All measurements are in microns (From Stock and Hunt, 2005).
Species TBL hermaphrodita1 (Schneider,1859) Andrssy, 1983 neopapillosa1 (Mengert in Osche, 1952) Andrssy, 1983 nidrosiensis2 (Allgn, 1933) Andrssy, 1983 papillosa2 (Schneider, 1866) Andrssy, 1976 valida2 (Sudhaus, 1974) Andrssy, 1983 1799 1509-2372 2227 1817-2449 1000-1750 1600-3400 NA FEMALE Tail Shape Elongate, conoid Elongate, conoid Cupola-shaped w/pointed tip Cupola-shaped w/pointed tip Cupola-shaped w/pointed tip Length 3-4 anal body widths long 3-4 anal body widths long 1.5-2 anal body widths long 1.5-2 anal body widths long 1.5-2 anal body widths long TBL Bursa shape MALE SpL

1585 1432-1771 900-1720 1200-2400 NA

Males are extraordinarily rare Well developed 1.5 times as long tail Small and narrow Well developed Well developed Twice as long as tail 1-1.5 times as long as tail NA

References: * Type species; NA= not available; SpL= spicule length; TBL= total body length. 1 After Hooper et al., 1999; 2 after Andrssy, 1983.

21

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

Key 3. Polytomus key to Heterorhabditis spp. microns (From Stock and Hunt, 2005).
Species TBL MBW

All data from original descriptions unless otherwise specified. Morphometrics are given in

Third Stage Infective Juvenile EP TL RF Hermaphrodite D% E% T shape indica-group IJ average size < 550 m NA NA conoid SpL GuL

Adults Male TREF

D%

BR

Female PAS

poinari

NA 350-410 418 332-499 528 479-573

NA 18-22 19 17-23 20 19-22

NA

NA

NP

NA 43-55 39 30-42 43 35-48

NA 24-32 18 14-21 21 18-23

NA

NA

NA

NA

taysaerae

90 74-113 98 88-107

55 44-70 101 93-109

NP

82 71-96 84 79-90

180 110230 94 83-103

conoid

122 100-146 106 78-132

NA

indica

NP

conoid

122 NA

7,8 do not reach the bursal rim 1 may be out of bursa 4, 7 outwards

bacteriophora-group IJ average size 550-700 m bacteriophora 588 512-671 551 497-595 572 528-632 685 570-740 654 588-700 23 18-31 20 18-22 22 20-24 27 22-30 28 24-32 103 87-110 97 91-103 111 104-116 112 94-123 102 81-113 98 83-112 90 83-97 76 68-80 102 87-119 107 99-117 NP 84 76-92 84 78-88 90 NA 80 70-84 77 60-86 112 103130 108 98-114 147 NA 108 103109 96 89-110 conoid 40 36-44 40 33-45 47 44-48 51 48-55 45 42-50 20 18-25 20 18-22 22 20-24 22 19-25 19 18-22 76 61-89 91 63-106 194 162-240 132 88-173 91 67-136 117 NA NA 4, 7 outwards NA P

baujardi

NP

conoid

brevicaudis zealandica marelata

NP NP NP

conoid conoid pipette-shaped

88 NA 118 NA 113 NA

NA 4, 7 outwards 4, 7 outwards 8 does not touch bursal rim

P V P

22

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

mexicana

578 530-620

23 20-24

102 83-109

99 91-106

NP

81 72-86

104 87-111

conoid

41 30-47

23 18-32

96 65-130

129 114-149

Arrange ment: 1,2,3,2 With 4, 7 outwards. Pairs 7-9 varaible

megidis-group IJ average size >700 m megidis downesi 768 736-800 879 669-1066 29 27-32 39 33-55 131 123-142 97 64-107 119 112-128 33 28-42 NP P 85 81-91 83 77-92 110 103120 169 129216 conoid blunt and mucronated 49 46-54 46 40-53 21 17-24 23 40-53 128 NA 122 NA NA 4,7 outwards 2,3 fused 4, 7 outwards 8 does not touch bursal rim P P

References: BR= bursal rays; D%= EP/oesophagus length x 100; E%= EP/TL x 100; EP= excretory pore; MBW= maximum body width; NA= information not available; PAS= post-anal swelling; RF= tail refractile spine; T=tail; TBL= total body width; TL= tail length; TREF= testis reflexion; V= variable.

23

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

Key 4. Key diagnostic features of mononchid genera considered in biological control (From Stock and Hunt, 2005).
Mononchus Bastian, 1865 Non-tuberculate Anterior half and forward Absent Mylonchulus Cobb, 1916 Non-tuberculate Anterior half and forward Small pair of teeth usually opposite base of dorsal tooth. Walls with 2-13 transverse rows of minute denticles. Iotonchus Cobb, 1916 Tuberculate Posterior half and forward Absent

Oesophago-intestinal junction Position/direction of dorsal tooth Subventral teeth or denticles

Key 5. Key diagnostic features of mermithid genera considered in biological control (From Stock and Hunt, 2005).
Diagnostic Features Cephalic papillae Labial papillae Oral opening Hypodermal cords Vagina shape Bursal sleeve Parasitic and Post-parasitic tail Eggs Agamermis Cobb, Steiner and Christie, 1923 6 Absent Terminal 6 S-shaped Absent With crater-like terminus Without byssi Mermis Dujardin, 1842 4 Present (2) Absent 6 S-shaped Absent With tail appendage With byssi Oesophagomermis Artyukhovsky, 1969 6 Absent Terminal or slightly shifted to ventral side 6 S-shaped Absent With small tail appendage Without byssi Romanomermis Coman, 1961 6 Absent Terminal 8 Pear-shaped Absent With tail appendage Without byssi Strelkovimermis Rubzov, 1969 6 Absent Terminal or slightly shifted to ventral side 6 S-shaped May be present With tail appendage Without byssi

24

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

E. Preparation of Nematodes for Identification


________________________________________________________________________
1. Obtaining nematodes A good stereomicroscope is essential for nematode identification and should have a range of magnification between 10 and 100X, a fairly flat field, and good resolution. Illumination by transmitted light should be as even as possible. Handling most nematodes in distilled water presents no problem; however, the use of a saline solution (i.e., Ringer's solution) is highly recommended to avoid osmotic shock. For living material, the nematodes can be mounted in Ringer's solution or water on a glass slide with supports and a cover-slide sealed with paraffin or nail polish. Perform dissections in a Petri dish with Ringer's solution and under stereomicroscope. Lift individual specimens out of the solution with the help of a handling needle of which there are many types: nylon toothbrush bristle, toothpick handling L-shaped needle, a dentist's probe, steel needle, a bamboo splinter, etc. For smaller nematodes, an eyebrow hair stuck on to the end of a mounted needle is very useful and does not damage the nematodes. Beginners will have some difficulty in picking up nematodes. It is recommended to use the lowest microscope magnification (to give good depth of focus and working distance) and to start with nematodes that are near the center of the dish. Place the needle underneath the nematode and lift up quickly so that the nematode is pulled through the meniscus. The time frame for doing the dissections may vary according to the different species/isolates. For steinernematids, dissections should be done 2-3 days after infection to recover first generation adults (males and females), and 5-6 days after infection for second generation adults. For heterorhabditids, dissections should be done 3-4 day after infection to recover first generation hermaphrodites, and during the 6-9 day after infection for second generation adults (males and amphimictic females). 2. Killing and fixing Heat.-Nematodes may be killed by heating in a water bath at 60oC for 2 min. Once the specimens are killed, add the fixative (TAF) to an amount equal of Ringer's solution. The temperature of the fixative and the Ringer's solution should be approximately 60oC. Leave specimensl for 24h in this mixture of 1:1 Ringer-TAF. Then, replace solution with pure TAF. Nematodes fixed in TAF may remain in this solution for several months/years. If you desire to make permanent mount, nematodes should be dehydrated following the Seinhorst method (see section 3) An improved method that kills and fixes the nematodes in one process is that of Seinhorst (1966). The specimens are collected in a very small drop (ca. 1 ml) of water in a Syracuse watchglass or similar deep concave vessel. The fixative (i.e., TAF) is heated to 100oC and an excess (3-4 ml) is added to the nematodes. The nematodes remain in this solution for 12 hr, and then the solution is replaced with double-strength fixative. Cooling.- Samples can be stored in the refrigerator (ca. 4oC) or in water with ice to relax the nematodes by cooling. Once the nematodes are relaxed, add fixative heated previously at 65oC. Allow the fixative to act for at least 1 day. Transfer the nematodes to a Syracuse watchglass with as little fixative as possible. Ringer's solution (Woodring and Kaya, 1988) - 9 g NaCl - 0.4 g KCl

25

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

- 0.4 g CaCl2 - 0.2 g NaH2CO3 -1 liter distilled H2O M9 Buffer (Brenner, 1974) - 3 g KH2PO4 - 6 g Na2HPO4 - 5 g NaCl - 1 ml MgSO4 (1M) - 1 liter distilled H2O Fixatives (Seinhorst, 1959, 1966; Southey, 1986; Woodring and Kaya, 1988) Numerous fixatives have been recommended for studying different features of nematode anatomy. Doublestrength fixatives are made up using half the amount of water indicated below. The commonly used fixatives are: TAF - 7 ml formalin (40% formaldehyde) - 2 ml triethanolamine - 91 ml distilled H2O F.A. 4:1 or F.A. 4:10 - 10 ml formalin - 1 or 10 ml glacial acetic acid - up to 100 ml distilled H2O 3. Preparing nematodes for permanent mounts The gonads and other structures of fixed nematodes may be obscured by the granular appearance of the intestine. Specimens can be cleared by processing to lactophenol or glycerin. Although both are good mounting media, nematodes will keep almost indefinitely if they are processed to glycerin. -Processing to glycerin Transfer fixed nematodes to a Syracuse watchglass containing 0.5 ml of Solution I (20 parts of 95% ethanol, 1 part of glycerin, 79 parts of distilled water) (Seinhorst, 1959). Place the Syracuse watchglass in a desiccator and add 95% ethanol to the desiccator so that the space

below the holding shelf is half full. Leave the watch-glass in this atmosphere for at least 12 hr in an oven at 35oC to allow slow evaporation of the ethanol from Solution I in the watchglass. Remove the watchglass from the desiccator. Then fill the watchglass with Solution II (5 parts of glycerin, 95 parts of 95% ethanol), place in a Petri dish which should be partially opened to allow slow evaporation of the ethanol, and maintain for 3 hr at 40oC. After processing the nematodes in pure glycerin, they are ready for mounting. -Mounting and labeling Aluminum double-coverslip slides (Cobb, 1917) are suitable for permanent mounts as they allow to view specimens from either side, are durable, and are less likely to crack during shipping or rough handling. If they are not available, good quality glass slides (76 x 25 x 1 mm) can be used. When using an aluminum slide, a square coverglass has to be slipped in the aluminum carrier between two pieces of cardboard. The edges of the carrier must then be pressed to hold the cardboard and the coverglass in place. Place a drop of glycerin in the center of the slide and place a round coverslip on top of the glycerine. Short pieces of glass fiber can be included to give support. Seal specimen slides with wax paraffin or a double ring of nail polish. A substance called Zut was previously recommended, but it is no longer commercially available. To seal specimen slides with paraffin, heat a 1.7 mm diameter metal tube with polished rim using a Bunsen burner or alcohol lamp. Press the tube into the paraffin and hold it against the glass to leave a ring of paraffin (the thickness of the ring should be in accordance with the size of the nematodes to be mounted). Place a drop of glycerin in the center of the ring and place a few nematodes in the bottom of the drop making sure that the nematodes are aligned and do not overlap. Place the slide on a hot plate and drop a coverglass on top as soon as the paraffin ring melts and immediately take the slide off the hot plate. Once the paraffin sets,

26

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

the slide is sealed and contains a small central area of glycerin and nematodes with a surrounding rim of paraffin. -Mounting cross sections The cephalic structures and the number of longitudinal chords are diagnostic characters for generic or specific determination of certain groups of nematodes (i.e., rhabditids and mermithids). These structures are best seen by making cross sections. Place the nematodes that have already been processed to glycerin in a drop of glycerin and cut into small sections (anterior end or midbody) with a razor blade or a small oculists scalpel. Using a needle, transfer the section to a drop of melted glycerin jelly in the center of a coverglass and orient in the proper position. Invert the coverglass and gently place on a slide with pieces of glass fiber to prevent the jelly from touching the slide. Seal the mount with paraffin or glycerin jelly. Recipe for hard glycerin jelly (Southey, 1986): - Soak 20 g of gelatin in 40 ml of distilled water for 2 hr; - Add 50 ml of glycerin and 1 ml of phenol; - Place in water bath (70-80oC) for 10-15 min and stir until the mixture is homogenous. 4. Preparation for scanning electron microscopy Scanning electron microscope is a very useful tool for the visualization and interpretation of certain nematode features, that cannot be appreciated with a light microscope but are important for their taxonomic identification. To prepare nematodes for viewing, place them in a water bath at 60oC for 2 min to kill them. Rinse nematodes three times in Ringers solution (pH 7.3) or phosphate buffer (pH 7.4) (5 min each change) and prefix in 8% glutaraldehyde (glutaraldehyde 25% EM grade, diluted in Ringers solution). Leave nematodes overnight in this solution. The next day,

rinse nematodes three times in Ringers solution (5 min each change), and once in water (5 min). Post-fix in 1% osmium tetroxide (OsO4) for 2 hr. After postfixation, rinse nematodes three times in water (5 min each) and dehydrate them using a series of ethanol washes (30, 50, 70, 90, 95 and 100%). The nematodes are then critical point dried with liquid CO2, mounted on SEM stubs, and coated with gold for 1 hr.

Plain agar plates for nematodes These plates are made at either 1% or 2% agar 10g per liter for 1%, 20g per liter for 2%. 1. Combine agar and 1 L of doubledistilled water in a large flask. 2. Autoclave for at least 30 minutes of heat. 3. Cool until comfortable to the touch. 4. Pour. 5. Allow to solidify and dry at room temperature overnight. 6. Re-bag and refrigerate. Baby Food plates for nematodes (Stock et al., 2001) The baby food seems to have some heat resistant spores in it. To eliminate these, a double autoclave technique called Tindallization is used autoclave agar, leave for 24 hours to allow bacteria to grow but not time enough for them to make resistant spores, then autoclave again. 1%: 10 g agar, 6 g Beechnut baby food (or any other 3 cereal baby mix) in 1 L distilled water. 1. Autoclave (at least 30 minutes of heat). 2. Leave at room temperature in the lab for 24 hours, then autoclave again. 3. Cool (water bath not necessary if swirled occasionally). 4. Pour 2%: 20 g agar, 0.5 g Beechnut baby food in 1 L distilled water. Autoclave (at least 30 minutes of heat), leave for 24 hours, then autoclave again, cool (water bath not necessary if swirled occasionally), and pour.

27

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

F. Atlas
_____________________________________________________________

28

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

General Morphology
Reproductive system Tail Egg Anterior region Stoma Amphid Oesophagus Nerve Ring Excretory pore Excretory cell Cuticle

Digestive system

From Maggenti, 1981

Transversal section
Muscles Epidermis Cuticle Intestinal Cavity Dorsal nerve

Excretory canal

Cuticle Glandular Cell General body cavity Ventral nerve

29

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

Excretory System

From Poinar, 1983 References: L: canal, G: excretory cell (gland), P: excretory pore; T: excretory canal A. H-shaped (e.g. Oxyuroids), B. I-shaped (e.g. Diplogasterids, Tylenchoids), C. Inverted Ushaped (e.g. Chromadoria), E. Single-cell (e.g. Chromadorids).

30

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

Digestive System Types of Oesophagi

A. Crenate (e.g. Enoplids), B. Multibulbar (e.g. Enoplids), C. Stichosome (e.g. Mermithids), D. Two-part oesophagus (e.g. Dorylaimids), E-F. Three-part oesophagus (E. Rhabditis, F. Diplogasterid). From Maggenti, 1981

Types of corpus

A. Enoplida, B. Dorylaimida, C. Rhabditida, D. Tylenchida. From Maggenti, 1981

31

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

Nervous System

Nerve Ring Nerve Ganglia

Nerve Canal

From Maggenti, 1981

Phasmid

From Maggenti, 1981

32

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

Male Reproductive System

testis

intestine

spermatocites

spermatozoa

Vas deferens

spicule

gubernacule

bursa

From Dropkin, 1989

33

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

Female Reproductive System

From Dropkin, 1989

34

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

G. Molecular Diagnostics / Phylogenetics


PHENOL-CLOROPHORM EXTRACTION Use: 1.7 ml Eppendorf tubes -Transfer nematodes to tubes and add the following: -500l of TH pH 8.0 -15l of 30% SDS -20l of proteinase K (1ng/ l) *note: if sample contains for example 100l (nematode suspension) add ONLY 400l of TE. This is adjust amount of TE added so final volume is 500l. -Incubate at 50C in water bath (incubation time varies according to the sample). -Check periodically for digestion. If this is not completed at the end of the day, add 15l more of proteinase K and leave overnight. -If digestion is completed place sample in freezer until next day. -The day after add 10l of RNAase to each sample. -Vortex and incubate at 37C for 1 hour. -Centrifuge at lowest speed (4 in our centrifuge) for 2 minutes. -Transfer supernatant to a new 1.7 eppendorf tube. Be careful when pipetting, avoid collecting the debris. -Add equal (to the amount of volume that is already in the tube) volume of phenol (in the hood) to each tube -Vortex briefly and spin (full speed for 5min. -Pipet supernatant carefully, (do not touch the phenol interface) and place it in a new eppendorf tube. -Discard the phenol in the 'phenol disposable container" in the hood. -Add equal volume of 24:1 clorophorm/isoamyl alcohol. -Vortex briefly and spin at full speed for 5min. -remove upper interface carefully and transfer to a new eppendorf tube. -Discard the isoamyl alcohol in the 'phenol disposable container" in the hood. -Centrifuge again the remaining clorophorm to be able to recover more upper interface (5min. at full speed) -Add Na acetate (to help precipitation) 10l per 100l. example if you have 400l in the tube add 40l of Na acetate (1M). -Add 100% ethanol to cover full volume of the tube and mix gently by hand. Place tubes in freezer and leave them overnight. -The following day spin in refrigerator's centrifuge at 4C for 10min at full speed. -Discard liquid and allow samples to dry in desiccator. -When samples are fully dried add 25l of TE to each tube. If pellet is big, add 50l of TE. Samples are now ready for spectrophotometry reading

CHELEX DNA EXTRACTION OF SINGLE NEMATODE You will need: Chelex resin (Sigma sells this as "Chelex 100"), Proteinase K, Sterile water 1) make 5% chelex soln (e.g. for a 10mL solution) A) place stir bar in 50mL conical tube held upright in a beaker B) add 0.5g Chelex resin into conical C) fill to 10 mL with sterile water This solution can be stored in the refrigerator for up to 1 month 2) Use a sterile razor blade to cut the end off of a P1000 tip to make the opening bigger (Chelex beads are too big to fit otherwise) 3) With Stir Bar going, mix up chelex soln (make sure chelex beads are spinning in the water) and take 20uL and add to tubes(make sure chelex beads are in there ). 4) Add 1 uL of proteinase K solution to each tube (20mg/mL). 5) Add nematodes to tubes, cutting each in half with a clean razor blade. 6) Incubate at 56 C for 1 hour. 7) Boil at 100 C for 8 min (100 C program in thermocycler).

35

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

8) Cool down for a few seconds (if using thermocycler bring down to 40 C for about 30 sec). 9) Vortex for about 30 seconds. 10) Use 4 uL of this in your PCR reactions, making sure no beads get in there. DNAZOL DNA ISOLATION (protocol for individual nematodes) 1. Place each live nematode in 100 mL digestion solution in a 0.5 ml Phoenix tube 100 mM Tris HCL pH 7.6 200 mL 200 mM NaCl 200 mL 0.5 M EDTA pH 8.0 400 mL 10 % Sarkosyl 200 mL Proteinase K (10 mg/ml) 20 mL MP3A water 980 mL Note Proteinase K should be made fresh usually 0.005 g/0.5 ml 2. Leave at room temperature for at least 0.5 hour to allow worm to digest solution 3. Digest overnight in 56 C water bath. 4. Heat kill proteinase with 95 C program in PCR machine (15 minutes) 5. Freeze and then thaw tubes 4 times 6. Centrifuge sample for 5 minutes at 10,000 rpm 7. Remove 95 mL of solution, leaving bottom 5 mL, and add it to 1 ml of DNAzol isolation reagent in a 1.7 ml tube 8. Add 4 mL Polyacryl Carrier solution, vortexing Carrier first 9. Mix tube by inverting 5 times 10 Add 0.5 ml 100 % ethanol and mix by inverting 10 times 11. Let sample sit at room temperature for 5 minutes 12. Pellet DNA by centrifuging at 7,000 rpm for 5 minutes 13. Pour off ethanol and then wash DNA twice with 800 mL 75% ethanol, spinning again if pellet breaks loose 14 Pour off final ethanol, then remove last of it with a pipetter 15. Allow visible ethanol to evaporate but do not allow pellet to dry 16 Resuspend in 6 mL lTE AGAROSE GEL 1. Make 1.3% gel: 40 ml of 1X or 0.5X TAE or TBE and 0.52g agarose Product: 7 mL water, 2 mL BPB dye, 1 mL vortexed product

Standard: 6 mL water, 2 mL BPB dye, 2 mL standard (Standard: 1 part Promega PhiX174/HaeIII marker, 2 parts lHind III marker) 2. Run 70-100 Volts for a couple of hours 3. Stain with 200 ml same running buffer and 35 mL Ethidium Bromide (1 mg/ml) on mixing machine for 15 minutes 4. Destain with 200 mL di water and 500 mL MgCl (0.1 M) on mixing machine for 20 minutes Notes: TBE resolves better than TAE but can precipitate and therefore stores less well. TAE runs slightly faster but disadvantage is slightly less resolution. PRIMERS (dilutions) PCR=10mM, Sequencing=3mM 1. Dilute with TE from stock for PCR primers (i.e. make 100 L of 10 mM); degenerate primers use at 20 mM for PCR. 2. Dilute PCR primers to make Sequencing primers (usually make 50 L:15 L of 10 mM PCR primer and 35 L lwater) no TE here as it inhibits cycle sequencing. For new dry primers, add 250 L lTE (Operon says pH 7), then determine concentration in mm based on pmoles given on oligo sheet. Sample calculation: DP585: Given 99126.16 pmoles on sheet and 250 L lTE DNTPs Purchased at 100 mM, so each one is diluted to 10 mM for PCR and combined (all 4) in equal amounts. There are already 1.7 L tubes with the diluted dNTPs in the same small blue box in freezer just aliquote equal amounts of these into a smaller tube or dilute more into the 1.7 L tubes if necessary.Enzymatic Treatment of PCR product

36

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

Cloning Procedures1. PCR product Preparation: 1.Spin filtration Use Ultrafree-MC spin filtration columns (Millipore 30,000 mwt filter) to clean up PCR product see protocol in box for more details. a. Wash product (all remaining) with 300 l of DEPC-treated water twice. Note that the filter must not be spun in excess of 5000 x g do 5,000 rpms for 5 minutes on the Hermle centrifuge . b. Remove product from filter by rinsing surface of membrane with DEPC-treated water using pipettor (but do not touch membrane surface with pipet tip). c. Adjust volume to ~20 l (or concentrate on speedvac if original gel band was light) 2. Ligation: 5 ul 2x ligation buffer (pre-aliquotted) 3 ul filtered PCR product (see step 1 above) 1 ul pGEMT vector in blue freezer box 1 ul T4 ligase Incubate ligation overnight at ~4C in refrigerator 3. Transformations Before beginning make sure LB plates are dry and at room temperature plates right from the refrigerator can be dried for 3 hours in the 37C incubator. a. Thaw competent cells on ice, mixing very gently b. Add 4 l of ligation to a 2 ml eppendorf tube - freeze remaining 6 ul. c. Add 50 l of thawed cells to eppendorf tube d. Gently mix (finger "vortexing") e. Place tube on ice for 90 minutes f. Heat-shock cells for 50 seconds at 42 C in water bath g. Place transformation on ice for 2 minutes h. Add 950 l SOC medium (at RT) Continued on next page i. Incubate transformation at 37 C for 1 hr (not longer) using rotating wheel in incubator j. Spread 50 l X-gal (20 mg/ml) onto room

temperature LB plates 1/2 hour before plating transformed bacteria. X-gal is kept in the 20 freezer in room 483. k. Plate 100 l of cells per plate (plate with IPTG, amp, & X-gal). Minimum of 2 plates per tranformation. l. Place tubes with remaining transformed bacteria in autoclave waste under sink in room 582. 4. Boil preps a. Pick piece of colony from master plate to 50 l of sterile water vortex. b. Boil 10 minutes in dry block (at at least 100C) (put brick on top of tubes) c. Spin tubes for 3 minutes d. Use 1 ul of supernatant as DNA source for 20 l PCR reaction use red label pipettors for adding supernatant as PCR product has been amplified by the bacteria. 5. Storing clones a. Use cryotubes. Grow cells in 800 l of LB with amp overnight (15 hr) on rotator wheel. b. Add 200 ul of 80% glycerol to grown cells. Mix by vortex. Freeze in 70C freezer. Transfer to liquid nitrogen tank and record placement on grid. 6. Broth culture/miniprep kit a. Put 5 ml LB with ampicillin broth in disposable culture tube. b. Inoculate with small piece of colony. c. Grow for 12 hours in shaker incubator at 37 C. d. Spin down tubes in large centrifuge for 5 minutes or until pelleted. e. Pour off broth, then pellet is ready to begin miniprep kit see protocol in kit. Media preparation LB plates 10g Tryptone 5 g Yeast Extract 5 g NaCl 1. Bring up to 1 L with MP3A water and pH to 7 with 5 N NaOH (about 200 L should do it). 2. Add 15 g agar, autoclave (at least 30 minutes of heat), cool in 50 C water bath. 3. Once cooled, add necessary

37

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

supplements: IPTG 100mM: add 5 L to 1 L LB agar for final concentration of .5mM. This is kept in the refrigerator in room 483. Ampicillin - add 2 tubes (25 mg/L) to 1 L of agar for final concentration of 75 mg/L. Aliquote and kept in the freezer 4. Pour plates. Leave at room temperature several hours to solidify. 5. Place in 37C incubator overnight to dry

and check for contaminant growth. 6. Once plates are dry and contaminantfree, they can be re-bagged and placed in the refrigerator. Before use bring plates to room temperature and spread 50 L of X-gal (20 mg/ml) at least 1/2 hour before spreading bacteria.

38

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

Schematic Representation of Genes considered for Nematode Phylogenetics and Diagnostics


Ribosomal DNA

3 2

Mitochondrial DNA

References: 1. Generic and above taxonomic hierarchies (family, etc); 2. Species and/or infraspecies levels; 3. Species level; 4-5. Species and/or infraspecies levels; 6. Infraspecific level

39

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

Origin of Nematode Parasitism in Insects and other Arthropods


Poinar, 1983)

(Modified from

40

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

Phylogenetic Framework of the Phylum Nematoda based on SSU rDNA Sequence Analysis
(Modified from Blaxter et al. (1998)

References: * Parasitic and/or associated with insect pests

41

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

H. References

_____________________________________________________________________________
Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77: 7194. Choo, H.Y., Kaya, H.K. and Kim, J.B. (1989) Agamermis unka (Mermithidae) parasitism of Nilaparvata lugens in rice fields in Korea. Journal of Nematology 21, 254-259. Choo, H.Y. and Kaya, H.K. (1994) Biological control of the brown planthopper by a mermithid nematode. Korean Journal of Applied Entomology 33, 207-215. Cobb, N. A. (1917). Notes on nemas. Contribution Science. Nematology No. 5. pp 117-128. Creighton, C.S. and Fassuliotis, G. (1983) Infectivity and suppression of the banded cucumber beetle (Coleoptera: Chrysomelidae) by the mermithid nematode Filipjevimermis leipsandra (Mermithida: Mermithidae). Journal of Economic Entomology 76, 615-618. De Ley, P. and Blaxter, M. (2002) Systematic position and phylogeny. In: Lee, D. L. (ed.) The Biology of Nematodes. Taylor and Francis, New York, pp. 1-30. Dropkin, V.H.(1989). Introduction to Plant Nematology. John Willey & Sons, Inc. NY, 304 pp. Hooper, D. J., Wilson, M.J., Rowe, J.A., Glen, D.M., 1999: Some observations on the morphology and protein profiles of the slug-parasitic nematodes Phasmarhabditis hermaphrodita and P. neopapillosa (Nematoda: Rhabditidae). Nematology, 1, 173-182. Kaya, H.K. and Stock, S.P. (1997) Techniques in insect nematology. In: Lacey, L. A. (ed.) Manual of techniques in insect pathology. Biological Techniques Series, Academic Press, San Diego, California, pp. 281-324. Lacey, L. A., Frutos, R., Kaya, H.K. and Vails, P. (2001) Insect pathogens as biological control agents: do they have a future? Biological Control 21, 230-248. Maggenti, A. R. (1981) General Nematology.Springer-Verlag, New York, pp. 372. Nickle, W.R. (1972) A contribution to our knowledge of the Mermithidae (Nematoda). Journal of Nematology 4, 113-146. Petersen, J.J. (1985) Nematode parasites. In: Chapman, H. C. (ed.) Biological control of mosquitoes. American Mosquito Control Association Bulletin 6, pp. 110-122. Poinar, G.O., Jr. (1975) Entomogenous Nematodes. E.J. Brill, Leiden, The Netherlands, 317pp. Poinar, G.O., Jr. (1983) The Natural History of Nematodes. Prentice-Hall, Inc., Englewood Cliffs, New Jersey, 323pp. Poinar, G.O., Jr. (1990) Entomopathogenic nematodes in biological control. In : Gaugler, R. and Kaya, K. H. (eds.) Taxonomy and biology of Steinernematidae and Heterorhabditidae. CRC Press, Inc., Boca Raton, Fl., pp. 2374. Seinhorst, J. W. (1959). A rapid method for the transfer of nematodes from fixative to anhydrous glycerin. Nematologica 4: 117128. Seinhorst, J. W. (1966). Killing nematodes for taxonomic study with hot F. A. 4:1. Nematologica 12: 178.

42

Diagnostics and Phylogenetics of Major Groups of Nematodes Considered in Biological Control Prepared by Dr. S. Patricia Stock - Locorotondo, Bari, Italy, June 12-13, 2005.

Southey, J. F. (1986). Laboratory methods for work with plant and soil nematodes. 6th Ed. Ministry of Agriculture, Fisheries and Food. Her Majesty's Stationary Office, London, UK. Stock, S. P. and Hunt, D. J. (2005). Nematode Morphology and Systematics. In: Grewal, P. S., Ehlers, R. U. and Shapiro-Ilan, D. I. (eds.) Nematodes as Biological Control Agents. CAB International (In Press).

Webster, J.M. and Thong, C.H.S. (1984) Nematode parasites of orthopterans. In: Nickle, W.R. (ed.) Plant and insect nematodes. Marcel Dekker, Inc., New York, USA, pp. 697-726. Woodring, J. L. and Kaya, H. K. (1988). Steinernematid and heterorhabditid nematodes: a handbook of techniques. Southern Cooperative Series Bulletin. 331. Arkansas Agricultural Experiment Station, Fayetteville, Arkansas. 30 pp.

43

Оценить