Entomopathogenic nematodes (EPN) belonging to the families Steinernematidae and Heterorhabditidae, are soil dwelling lethal parasites of insects that are used for inundative, augmentative or inoculative biological control of crop pests (Bedding et al., 1993; Gaugler and Kaya 1990; Kaya 1990; Kaya and Gaugler 1993; Parkman and Smart 1996). Due to their high biocontrol potential and eco friendly nature, the research on these nematodes is now going on in several laboratories worldwide and several new strains have been isolated. Their ever increasing importance in integrated pest management demands precise identification of species and their strains for not only pursuing further research on them but also for patenting and regulatory purposes, which require a sound biosystematics base. Till date, there are two genera listed under Steinernematidae - Steinernema and Neosteinernema, with 32 species in the former and only one species in the latter. Under Heterorhabditidae, there is only one genus - Heterorhabditis with only 8 known species. Of these, only two species have been reported from India: Heterorhabditis indica by Poinar et al.,(1992 ) and Steinernema thermophilum by Ganguly and Singh (2000). S. thermophilum, being heat-tolerant, may prove to be useful for incorporating in integrated pest management in tropical and subtropical parts of the world. Besides, several indigenous strains of Steinernema as well as Heterorhabditis have been isolated of which a few have been identified but many more are yet to be identified. In fact, most of the workers engaged in EPN research in India find it hard to identify the species due to lack of adequate taxonomic expertise in the extraction of adults from the insects, processing techniques, and non-availability of compiled literature. The taxonomic information on EPN was earlier compiled by Poinar (1990) and Hominick et al.,(1997). This chapter attempts to discuss briefly about the biodiversity and systematics of EPN, various taxonomic approaches, and diagnostic characters, along with compendia to the species, which may serve as an aid for species identification. The progress of EPN research carried out in the country has also been critically reviewed, identifying the gaps in knowledge and suggesting the future line of research.

Taxonomic Developments

Steinernematidae: The first steinernematid nematode, Aplectana kraussei was described from Germany by Steiner (1923). Travassos (1927) erected the genus Steinernema for this species. In 1929, Steiner proposed a new genus Neoaplectana and described it as N. glaseri, but did not give clear differences between this genus and Steinernema. Subsequently, more species were described under Neoplectana because it was more completely defined with the type species N. glaseri, while Steinernema remained monotypical with only S. kraussei. Both the genera remained grouped under the family Oxyuridae. Filipjev (1934) created the subfamily Steinernematinae and placed it under Anguillulidae under order Anguillulata. Chitwood & Chitwood (1937) upgraded the subfamily Steinernematinae to familial rank Steinernematidae. An examination of original type specimens of S. kraussei revealed no differences with Neoaplectana with regard to number and arrangement of head papillae and Neoaplectana was subsequently synonimized with Steinernema ( Wouts et al., 1982). This action created some confusion since the type specimens were not in good condition, and it became very important to search for some isolates, which could fit into Steiner's original description of A. kraussei. In order to avoid further confusion, Poinar (1990) designated S. kraussei as species inquirendiae and the name kraussei as nomen dubious, while S. glaseri as the type species. Mracek et al.(1991) discovered the type locality of S. kraussei in the Egge Mountains in Westphalia, Germany, and studied the morphological detail of the topotype specimens which was in conformity with S. kraussei of Steiner ( Mracek, 1994 ). Resultantly, S. kraussei was proposed as the type species of Steinernema. In view of the increasing awareness about the biocontrol potential of these nematodes, search for new isolates from different parts of the world intensified during the last one decade which has led to the description of several new species and their reports from different regions. There were only 9 valid species of this genus till 1990 which has now gone up to 32 during the last 12 years, and several strains reported from almost all the continents excepting Antarctica. Most of the species have been described after isolating the specimens from soil using insect bait while a few have been isolated from natural insect host. Table 1 depicts the list of valid species along with their type hosts and localities.

Heterorhabditidae: The history of heterorhbditids dates back to 1976 when Poinar first proposed the family Heterorhabditidae and the genus Heterorhabditis with the type species, H. bacteriophora isolated by him from Heliothis punctigera from South Australia. The H. bacteriophora was differentiated from other members of Rhabditoidea by possessing a vestigial valve in basal bulb, a reduced stoma, and dauer stages capable of entering the healthy insects, and exhibiting heterogony i.e producing hermaphroditic females in first generation and amphimictic females and males in the second generation. Aside from the members of Neoplectana, it was the only member known to serve as a vector for bacterial disease of insects. He also transferred Rhabditis hambletoni of Preira, 1937 to this genus. Later, N. hoptha Turco,1970 was also transferred to Heterorhabditis by Poinar (1979). Simultaneously, Khan et al.,(1976) proposed a new genus Chromonema with its type species, C. heliothidis, described from Heliothis zea from North Carolina, differing from neoplectanids in having peloderan bursa and straight to slightly curved spicules; production of hermaphroditic females in first generation and amphimictic females and males in the second generation and cadavers with brick red colour exhibiting bioluminescence in the dark. Chromonema was grouped under Steinernematidae after amending its diagnosis. They also studied the type specimens of Rhabditis hoptha and R. hambletoni and found these to closely resemble C. heliothidis. Since the two independently proposed genera Chromonema and Heterorhabditis shared the same characters, Poinar et al.(1977) proposed Chromonema as a synonym of Heterorhabditis and C. heliothidis became H. heliothidis. In 1979, Wouts identified a population of H. heliothidis from New Zealand, which was later found to have 41% dissimilarity from NC strain of H. bacteriophora, based upon biochemical data (Akhurst,1987). Detailed studies on its morphological features revealed it to be a distinct species and named as H. zealandica by Poinar (1990). Poinar et al., (1987) described H. megidis from infected Japanese beetle larvae, Popilla japonica from Ohio, which was different from H. bacteriophora in the characters of infective juveniles and by having pseudopeloderan bursa in males. The symbiotic bacteria present in the intestine of IJs was identified as Xenorhabdus luminescens. Thereafter, 6 more species were described: H. indica by Poinar et al., 1992; H. argentinensis by Stock,(1993); H. brevicaudis by Liu,(1994); H. hawaiiensis by Gardner et al., (1994); H. marelatus by Liu & Berry, (1996); and H. hepialius by Stock et al., (1996), the last one was found to be a synonym of H. marelatus. Due to the lack of adequate details, H. hoptha and H. hambletoni were considered as species inquirendiae. Unlike steinernematids, very few species have been described in this group which indicate that there is not much morphological and genetic biodiversity in heterorhabditids. Presently, there are only 8 valid species of Heterorhabditis.

Families of Insect Parasitic nematodes

Systematic position
Phylum:Nematoda Rudolphi, 1808
Class:Secernentea von Linstow, 1905
Order:Rhabditida (Orley, 1880) Chitwood, 1933
Suborder:Rhabditina (Orley, 1880) Chitwood,1933
Superfamily: Rhabditoidea (Orley,1880) Travassos, 1920
Family:Steinernematidae Chitwood and Chitwood, 1937
Type genus:Steinernema Travassos, 1927
Syn. Steineria Travassos,1927 nec Steineria Micoletzky, 1922
Neoplectana Steiner, 1929
Type species: S. kraussei (Steiner,1923) Travassos,1927
Syn. Aplectana kraussei (Steiner,1923) Travassos, 1927
Other species: 31 valid species ( as listed in Table 1)
Other genus: Neosteinernema Nguyen and Smart,1994
Type and the only species: N. longicurvicauda Nguyen and Smart,1994

Family: Heterorhabditidae Poinar, 1976
Type and the only genus: Heterorhabditis Poinar, 1976
Syn. Chromonema Khan, Brooks & Hirschmann, 1976
Type species:H. bacteriophora Poinar, 1976
Syn. Chromonema heliothidis Khan et al., 1976
Heterorhabditis heliothidis (Khan et al.,1976) Poinar et al., 1977
Other species:7 valid species.
Family Heterorhabditidae Poinar, 1976

Diagnostic characters: (Rhabditida. Rhabditoidea. Obligate parasites of wide range of insects. Adults: Stylet absent. Head truncate or slightly rounded, having six distinct lips which may be fused at base; each lip with a single labial papilla; two additional papillae at the base of each submedian lip; lateral lip with a single cephalic papilla and a circular amphidial opening. Cheilorhabdions present as a refractile ring in anterior part of stoma. Posterior part of stoma collapsed , with reduced pro-, meso, and metarhabdions. Base of stoma surrounded by oesophagous. Oesophagous with wide and cylindrical procorpus, followed by narrow isthmus, and then expanding into distinct basal bulb with reduced valve plates. Nerve ring distinct, surrounding isthmus in females and basal bulb in males. Excretory pore always located posterior to nerve ring in all the stages. Females: Hermaphrodites with sperms in proximal portion of ovotestes and functional vulva. Amphimictic females with didelphic amphidelphic reflexed gonad. The reflexed portion long, extending past the vulval opening. Sperms in proximal part of oviduct. Vulva functional only for mating, but otherwise non-functional due to ovoviviparous nature. Mated females often with a mucoidal substance (mating plug) around the vulva. Tail pointed with postanal or anal swelling. Rectal glands present. Males: Testis single and reflexed. Spicules paired and separate, nearly straight. Gubernaculum present. Bursa present, open, peloderan or weekly leptoderan, supported by nine pairs of genital papillae.
Infective juveniles: Third stage juvenile often inside the second stage cuticle. Body tapers more in the posterior region to a long acutely pointed tail. Head with armature on the dorsal side (hook or spine) sometimes opposed by small subventral spine. Mouth and anus closed. Oesophagous narrow with a weak basal bulb having weakly developed valve plates. Cuticle with paired longitudinal double lines, while second stage ensheathing cuticle with numerous longitudinal ridges. Excretory pore posterior to nerve ring. IJs develop into hermaphrodites.
Bionomics: Heterorhabditids are 'cruisers' in their foraging behaviour. The third stage juvenile is the infective stage which carries the symbiotic bacteria, Photorhabdus luminescens in its intestine. After invading the host, the bacteria are released in the hemolymph where they multiply and the host dies within 24-48 h due to septicaemia. The cadavers turn mostly pink, red, purple, orange, yellow and sometimes green in colour. Heterorhabditid-infected insects luminesce in dark due to the characteristic nature of mutualistic bacteria. Infective juveniles feed on the bacteria as well as degraded contents of hemocoel and develop into hermaphrodites in first generation, within 3-4 days after infection (DAI). The progeny of hermaphrodites develop into second generation amphimictic females and males within 6-9 DAI. After completing 2-3 generations, the nematodes emerge from the cadaver as third stage juveniles which are ready to infect another healthy insect. These can be cultured in vivo using the insect host, or in vitro on symbiotic bacteria on an artificial medium and have very high biocontrol potential against the insect pests of crops.
Type genus:Heterorhabditis Poinar, 1976
Diagnosis:Rhabditida, Rhabtoidea, Heterorhabditidae. (Characters of the family).
Type species: Heterorhabditis bacteriophora Poinar, 1976
Syn. Chromonema heliothidis Khan, Brooks and Hirschmann, 1976
Heterorhabditis heliothidis (Khan, Brooks and Hirschmann, 1976)
Poinar, Thomas and Hess, 1977
Other species:
H. argentinensis Stock, 1993
H. brevicaudis Liu, 1994
H. hawaiiensis Gardner, Stock and Kaya, 1994
H. indica Poinar, Karunakar and David, 1992
H. marelatus Liu and Berry, 1996
Syn. Heterorhabditis hepialius Stock, Strong and Gardner, 1996
H. megidis Poinar, Jackson and Kline, 1987
H. zealandica Poinar, 1990
Species inquirendae
H. hoptha (Turco, 1970) Poinar, 1979
(= Neoplectana hoptha Turco, 1970)
H. hambletoni (Pereira, 1937) Poinar, 1976
(= Rhabditis hambletoni Pereira, 1937)

Compendium and Key to the species

A compendium of valid 8 species of Heterorhabditis has been prepared on the basis of morphometric characters of infective juveniles (Table 4) and first generation males. Hominick et al., (1997) provided a key to the species of Heterorhabditis wherein they used the character "presence or absence of rostrum in spicules" which may not be easily seen by novice taxonomists. Therefore, following key to the species of Heterorhabditis is proposed with some modifications.