Nematode Biology

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The parasitic cycle of nematodes is initiated by the third-stage IJs. These non-feeding juveniles locate and invade suitable host insects through natural body openings (i.e., anus, mouth, and spiracles). Once inside the host, nematodes invade the hemocoel and release the symbiotic bacteria that are held in the nematode's intestine. The bacteria cause a septicemia, killing the host within 24-72h. The IJs feed on the rapidly multiplying bacteria and disintegrated host tissues. About 2-3 generations of the nematodes are completed within the host cadaver. When food reserves are depleted, the nematode reproduction ceases and the offspring develop into resistant IJs that disperse from the dead host and are able to survive in the environment and to seek out new hosts. From a commercial standpoint, the production of a durable infective stage and the symbiotic association with a lethal bacterium are the two most attractive features of steinernematid and heterorhabditid nematodes. The IJs are capable of tolerating stresses fatal to other developmental stages, and therefore can be effectively formulated and preserved for several months and shipped for application. The IJs are produced when conditions inside the insect cadaver are no longer suitable for further reproduction, and are the only stage in the nematode life cycle that leaves the host to infect a new host. The ventricular portion of the intestine of the steinemematid IJ is specifically modified for storage of symbiotic bacteria, and is called an intestinal vesicle. In the infective stage of heterorhabditid nematodes, symbiotic bacteria are located in the esophagus and in the ventricular portion of the intestine. The symbiotic association of EPN with specific bacteria facilitates rapid mass production of the nematodes (bacteria serve as food) and successful pathogenicity. Although axenic nematodes (nematodes without bacteria) may occasionally cause host death, they do not generally reproduce. Furthermore, bacteria alone are incapable of penetrating the alimentary tract and cannot independently gain entry to the host's hemocoel. Thus, nematodes act as vectors to transport the bacteria into a host within which they can proliferate, and the bacteria create conditions necessary for nematode survival and reproduction within the insect cadaver. All species of Steinernema are associated with bacteria of the genus Xenorhabdus and all Heterorhabditis nematode species are associated with Photorhabdus bacteria. Each nematode species has a specific natural association with only one bacterial species; though any one bacterial species may be associated with more than one nematode species (Table 1). The specificity of association between nematodes and bacteria operates on three levels 1) Provision of factors that enhance Infective Juveniles (IJ) recovery from the dauer phase 2) Provision of essential nutrients for the nematode by the bacterium and 3) Retention of the bacterium within the intestine of the non feeding IJ. Infections by EPN can be recognized by various signs. Soon after death the cadavers turn flaccid and start changing colour. In the case of wax moth larvae and depending upon nematode species, steinernematid -killed insects turn to various shades of brown, ochre whereas heterorhabditid killed insects turn red, brick red, purple orange, yellow and sometimes green. The colour of the cadaver is attributed to the associated bacterium; especially Photorhabdus that have pigments .The steinernematid -killed insects stay flaccid throughout the nematode development, whereas the heterorhabditid-killed insects become less flaccid. If the cuticle is transparent, nematodes are visible inside the cadaver. The cadavers do not putrefy, and even when dissected, do not have a putrid odour. The body contents lose their integrity over time but never liquefy. In heterorhabditid-killed insects, the body contents become ropy.

DIFFERENCES BETWEEN STEINERNEMATIDS AND HETERORHABDITIDS
CHARACTERS STEINERNEMATID HETERORHABDITID
Secondary stage cuticle Easily lost Good retention
Secondary stage cuticle visibility Loosely fitted, Easily seen Tightly fitted, Difficult to see
Develops to Male or female Hermaphrodite
Bacterial symbionts Modified ventricular intestine Through the intestine
Luminescence No Yes
Colour of dead larvae Ochre, black Red, pink
Reproduction Amphimictic Hermaphroditic- 1st & Amphimictic - 2nd generation
Bursa in male No Yes

BIOLOGY/LIFE CYCLE

Steinernema and Heterorhabditis (Rhabditida) are symbiotically associated with bacteria of the genera Xenorhabdus and Photorhabdus within the Enterobacteriaceae in the gamma subdivision of purple bacteria,respectively. Each nematode species has a specific association with one bacterium species. Like other nematodes of the Order Rhabditida, Steinernema and Heterorhabditis spp. form dauer (enduring) juveniles (DJs), which are morphologically and physiologically adapted for long term survival in the soil environment. DJs can be isolated from almost all habitats where soil insects occur. Whereas the few Heterorhabditis species are distributed all around the world, the majority of the Steinernema species seem to be restricted to certain geographical regions and other species, e.g. S.feltiae, are widely distributed. The nematode dauer juveniles (DJ) carry between 0 to 250 cells of their symbiont in the anterior part of the intestine. The symbiotic bacteria are released into the haemolymph after penetration of the DJ into a suitable host insect. Inside the DJ the bacteria are well protected against detrimental conditions in the soil. Neither Xenorhabdus nor Photorhabdus spp. have ever been isolated from soil environments. Accordingly, this phoretic relation seems to be of vital necessity for the associated bacteria. They totally depend on transmission by the DJ into a sterile environment like the insect's haemocoel, as they lack any means for survival in the soil environment or invasion of insect's haemocoel without the help of the DJ. Whereas Steinernema spp. can kill insects even without their symbiotic bacteria, Heterorhabditis spp. lack insect pathogenicity in the absence of P. luminescens. Host-finding behaviour of DJs can differ within a population and can also be species specific. "Hunters" are highly mobile and a large proportion of their population tends to actively seek for suitable hosts. In populations of S. glaseri and Heterorhabditis spp. the majority of the individuals show this character. The sit-and wait "ambushers" often attach to soil particles and nictate. Waiting for an insect to pass by and then attack. S. carpocapsae is a species with this behaviour. Penetration of the host insect occurs via natural openings or directly through the insect cuticle. There are indications that the penetration process is supported by proteolytic factors produced by the exsheathed dauer juvenile. Upon reaching the haemocoel the DJ is recognized as non-self and insect defence mechanisms can eliminate the DJs through encapsulation. Defence mechanisms against the bacteria also have been described. Providing the insect's defence mechanisms fail to eliminate the nematode-bacterium complex, the insect dies 2-4 days after infection. Akhurst & Dunphy (1993) and Simoes & Rosa (1996) have summarized the current knowledge on the pathogenicity mechanisms of the nematode-bacterium complex and the interactions with the defense system of host insects. Once established inside the cadaver, the bacteria proliferate and produce suitable conditions for the nematode to grow and reproduce. The nematodes feed on cells of their symbiont and host tissue. Without the presence of the symbiotic bacteria in the insect cadaver the nematodes are unable to reproduce. Infective DJs of Steinernema develop to amphimictic adults and Heterorhabditis spp. to self-fertilizing hermaphrodites. Their offspring either develop to DJs or to a F1 adult generation. Another adult generation IJs (F2) is usually not developed. Instead, in response to depleting food resources, DJs are formed, Two to three weeks after colonisation of the host insect, the DJs leave the cadaver searching for new target insects in the soil.

Abstracts:

1. Glazer-I; Koltai-H; Zioni(Cohen-Nissan)-S; Segal-D1994. Life cycle and reproduction in Heterorhabditis. Genetics-of-entomopathogenic-nematode-bacterium-complexes.-Proceedings-and-National-Reports-1990-1993,-St.-Patrick's-College, -Maynooth,-Co.-Kildare,-Ireland. 1994, No. EUR 15681 EN, 80-89; Proceedings of a Symposium and Workshops held at St. Patrick's College, Maynooth, Co. Kildare, Ireland on Oct. 23-27, 1993
AB: Using Galleria mellonella larvae at 25°C the duration of the life cycle of H. bacteriophora strain HP88 from egg hatching to egg hatching was 96 h. Juvenile development lasted 48 h, with the duration of each juvenile stage ranging from 8-12 h. Under crowded conditions development proceeded to the infective juvenile (IJ) stage instead of the J3 stage. Duration of the life cycle and proportion of various developmental stages was similar in in vitro and in vivo cultures. When in vivo or in vitro development was initiated from the IJ stage, only hermaphrodites developed in the first generation and males appeared only in the second generation. The average number of progeny/ hermaphrodite was 243 ± 98. Forty hours after egg hatching 2 types of juveniles were evident in the second generation of the wild-type or mutant population cultured in vitro. Half of the second generation had propagated to the J4 stage with discernible reproductive systems. All other individuals were less developed and had no identifiable reproductive system. The mode of reproduction of these 2 types of individuals were examined.

2. Koltai-H; Glazer-I; Segal-D. Reproduction of the entomopathogenic nematode Heterorhabditis bacteriophora Poinar, 1976: hermaphroditism vs amphimixis. Fundamental-and-Applied-Nematology. 1995, 18:1,55-61.
The present study was aimed at elucidating the mode of fertilization (self vs cross) in 2nd generation non-male adults of the Heterorhabditis bacteriophora strain HP88. For this purpose dumpy mutants (Hbdpy-1 and Hbdpy-2) were used as genetic markers. Forty hours after eggs hatching two types of juveniles were evident in the 2nd generation of either the wild-type or the mutant populations cultured in vitro: half of the 2nd generation individuals developed to the 4th developmental stage (J4) with discernible reproductive systems. The other individuals were, on average 1.4-2 times shorter and 1.6-3 times thinner (p‹0.05, t test) than the above described "normal" J4. They were less developed than the J4 type and had no identifiable reproductive system. Among 550 of the J4 type juveniles (either wild-type or dumpy) that had been individually transferred to culture plates, only 9 (i.e. 1.8%) gave rise to progeny. However, when dumpy non-male adults, originating from J4 type juveniles were crossed to wild-type males, 30-71% of them gave rise to progeny all of which were wild-type, indicating that reproduction occurred solely by cross-fertilization. These non-male adults were termed "females". Among 105 smaller-type juveniles which had been individually transferred to culture plates, 80% reproduced indicating a high rate of self-fertilization i.e., a high proportion of hermaphrodites. The smaller type juveniles were termed "HJ" (H for hermaphrodite). When dumpy HJ type juveniles were crossed with wild-type males, 70% (n = 30) gave rise to progeny. Each successful cross yielded both dumpy (46%-69%) and wild-type (31%-54%) progeny, indicating reproduction by self as well as cross-fertilization, respectively. The importance of the co-existence of these two reproductive strategies and their implication to genetic studies are discussed.

3. Poinar-GO Jr. Description and biology of a new insect parasitic rhabditoid, Heterorhabditis bacteriophora n.gen., n.sp. (Rhabditida; Heterorhabditidae n.fam.). Nematologica. 1975 publ. 1976, 21: 4,463-470.
AB: Heterorhabditis bacteriophora n.g., n.sp. is described from Heliothis punctigera from Brecon, South Australia. Heterorhabditidae n.fam., proposed under Rhabditoidea for Heterorhabditis is recognized by a combination of these characters: 6 lips, vestigial stoma with reduced or modified rhabdions; terminal pharyngeal bulb lacking bulb flaps but having a haustrulum with thickened lining; heterogonic cycle with both hermaphroditic and dioecious females; males with a well developed papillate bursa and dauer stages capable of entering the haemocoel of healthy insects. Rhabditis hambletoni Pereira, 1973 is transferred to Heterorhabditis. Aside from Neoaplectana spp., H. bacteriophora is the only nematode known as a vector for a bacterial disease of insects. The infective stage juveniles of this nematode carry a specific bacterium in their intestines which is released after the parasites enter the body-cavity of a healthy insect. The bacteria kill the insect in 48 hours and the juveniles develop into hermaphroditic females that produce young which develop into males and females. The latter mate and produce juveniles that develop into infective stages, which leave the cadaver and search for a new host. The infective stage of H. bacteriophora can invade and kill larvae of Galleria mellonella in 48 hours and it can also destroy larvae of Culex pipiens.

4. Gudkov-II. A study of the biology of the insect nematode Heterorhabditis bacteriophora. Gel'minty-nasekomykh. 1980, 34-37.
AB: Giant hermaphrodite females of H. bacteriophora developed 4 to 5 days after the death of their experimental host, Tenebrio molitor; larvae appeared on days 7 to 8 and adults of the 2nd (sexual) generation were observed on days 8 to 9; 2nd-generation L3 were formed by day 11 or 12. These parasitic larvae could remain latent, in the absence of a host, for a long time. Altogether the development (from the death of the insect to the appearance of L3) took 16 to 17 days at 20 to 22 deg C. The introduction of foreign micro-flora by the nematode into the insect caused putrification of the insect and death of the nematode, this was rapid and total when infection was introduced into the haemocoel directly but if infection was introduced via the cuticle or intestine, part of the 2nd generation accomplished their life-cycle. The effects of other conditions (such as the length of time during which the culture was kept, the substrate used) on the life-cycle, micro-flora and yield of nematodes are also described. Combined infection with H. bacteriophora and Neoaplectana carpocapsae or N. feltiae was noted and such cultures survived successfully for 4 months. Introduction of Rhabditidae, Diplogasteridae or Acarids killed the culture in 1.5 to 3 months.

5. Kaya-HK; Burlando-TM. Development of Steinernema feltiae (Rhabditida: Steinernematidae) in diseased insect hosts. Journal-of-Invertebrate-Pathology. 1989, 53:2, 164-168.
AB: The interaction between the entomophilic nematode Steinernema feltiae [Neoaplectana carpocapsae] and a nuclear polyhedrosis virus (NPV) or Bacillus thuringiensis subsp. kurstaki in a common host (larvae of Galleria mellonella, Spodoptera exigua and Bombyx mori for the 3 pathogens, respectively) was investigated. N. carpocapsae reproduced successfully in 68.3% of moribund hosts infected with NPV, N. carpocapsae infectives retained sufficient NPV to infect neonate larvae of nematodes from NPV-infected hosts when fed whole or macerated to neonate S. exigua larvae caused 44 and 58% NPV infections, respectively. On the other hand, N. carpocapsae did not produce progeny in Bacillus thuringiensis-infected hosts, except in a few cases. Those hosts which had a dual infection had B. thuringiensis infection in the anterior part and N. carpocapsae infection in the posterior part of the body. In general, B. thuringiensis-killed insects were not satisfactory hosts for N. carpocapsae.

6. Nguyen-KB. A new nematode parasite of mole crickets: Its taxonomy, biology and potential for biological control. Dissertation-Abstracts-International-B-Sciences-and-Engineering. 1988 (publ.) 1990, 50:7, 2747; Abstract of thesis, 167 pp.
AB: A new steinernematid nematode parasite of mole crickets [Gryllotalpidae] was collected from Uruguay, South America. The nematode does not fit any nominal species of the genus Steinernema and is herein described as a new species. The life cycle and sex ratio of the nematode is influenced by temperature. At 10 to 15°C the life cycle is not completed, at 20°C the cycle takes 12 days to complete, at 24°C, 10 days and at 30°C, 8 days. At 15-24°C the number of females in the population is greater than the number of males, but at 30°C the reverse occurs. The new species does not reproduce well, if at all, in larvae of the wax moth, Galleria mellonella, which is a universal host for all other species. Its host range appears to be much narrower than that of other species, with a penchant for mole crickets. When released in the field in North Florida, the nematode became established, has spread out from the original release sites, and continues to kill mole crickets after 3 years 6 months. When released on the soil surface, the nematode moved down and killed mole crickets placed 10 cm below the soil surface. The nematode survived in the soil for 10 weeks and retained its ability to kill mole crickets. These attributes make it a very good candidate for the biological control of mole crickets imported accidentally to Florida. In a survey of nematodes associated with mole crickets, the following genera were found: Binema, Cameroonia, Cruznema, Diplogaster, Mesorhabditis, Pulchrocephala, Steinernema and Talpicola.

7. Ghally-SE; Kamel-EG; Nasr-NM. Comparative studies on the development of the entomogenous nematode Steinernema feltiae Filipjev on Spodoptera littoralis (Boisduval) and Musca domestica Linnaeus. Journal-of-the-Egyptian-Society-of-Parasitology 1991, 21:3, 685-698.
AB: Comparative studies were made under laboratory conditions (26°C) on the development of Steinernema feltiae in different stages of Spodoptera littoralis and Musca domestica. It was shown that the rate of development was faster in S. littoralis than in M. domestica. When the timing of various life-cycle stages of Steinernema feltiae was examined, there were significant differences between the 2 hosts. The number of giant forms observed corresponded to the number of invasive larvae.

8. Molyneux-AS. The biology and ecology of the entomopathogenic nematodes Heterorhabditis spp. (Heterorhabditidae) and Steinernema spp. (Steinernematidae). [Thesis abstract]. Journal-of-the-Australian-Entomological-Society. 1985, 24:2,86.
AB: The influence of a number of key environmental factors on the behaviour and survival of various species of Heterorhabditis and Steinernema was studied in relation to the soil habitat and the presence or absence of insect hosts. In the absence of hosts, Heterorhabditis sp. D1, H. heliothidis strain T327 and S. feltiae Agriotos strain [Neoaplectana carpocapsae] showed an inverse relationship between survival time and temperature, whereas S. glaseri [N. glaseri] strain KG survived for many months at each temperature tested; infective juveniles of N. glaseri strain KG became quiescent in sand when insect hosts were not available but those of the other nematode species and strains did not. Infection of 3rd-instar larvae of Lucilia cuprina by Heterorhabditis sp. D1 and N. glaseri occurred over a wide range of soil moisture potentials. All of 13 species and strains of nematodes tested were able to kill all of 9 species of insects, but the degrees of infectivity varied. The relationship between nematode development rate and temperature was linear. For the first time, theoretical threshold temperatures for development were calculated that gave a close approximation to the observed lower temperature limits for development.

9. Johnigk-SA; Ehlers-RU 1999. Juvenile development and life cycle of Heterorhabditis bacteriophora and H. indica (Nematoda:Heterorhabditidae). Nematology, 1:3, 251-260; 18 ref.
AB:H. bacteriophora and H. indica were cultured in vitro in monoxenic liquid cultures. Nematodes were examined for morphological characters which can be used to determine the sexes in all juvenile stages. When hatching from the egg, no sex-specific characters could be distinguished. Twelve hours later sex determination resulted in male phenotypes which were identified by the asymmetric shape of the primordial testis. Female phenotypes, with symmetrical primordial gonads, were observed 12 h later. Of the eggs laid by the parental hermaphrodite 57% developed to amphimictic adults. The other individuals were determined to become automictic dauer juveniles which further develop to hermaphrodites. The pre-dauer J1 was the last of the first juvenile stages to be determined. They moult to the pre-dauer second stage juvenile (J2D) which is distinguished from the other J2 stages by a thin and spindle-like shape, elongated pharynx and needle-like tip of the tail. The late JD2 stage is immobile, pharyngeal pumping ceases and intercalated fat reserves make it appear darker then the amphimictic J2. The further development to adults is described and the occurrence of the different stages in liquid culture documented.

10. Boff-MIC; Wiegers-GL; Gerritsen-LJM; Smits-PH 2000. Development of the entomopathogenic nematode Heterorhabditis megidis strain NLH-E 87.3 in Galleria mellonella. Nematology. 2000, 2:3, 303-308; 30 ref.
AB: Increasing densities of Heterorhabditis megidis strain NLH-E 87.3 infective juveniles (IJ) affected invasion, reproduction, length and time to first emergence of the nematodes in larvae of the greater wax moth, Galleria mellonella. Although the number of nematodes that invaded the host increased with increasing dose, percentage of invasion declined. The number of progeny produced per host initially increased with dose. The highest production of IJ per cadaver was reached at a dose of 300 IJ per host, at that dose 62±3.4 IJ were established per cadaver. Production decreased again significantly at higher densities. The smallest IJ were produced at a dose of 1000 IJ per host and the largest at a dose of 300 IJ per host. Time to first emergence of juveniles was generally shorter when the number of IJ inoculated was large (300-3000 IJ/host).

11. Elawad-SA; Abbas-MS; Hague-NGM. The establishment, reproduction and pathogenicity of a new species of Steinernema from the Sultanate of Oman in Galleria mellonella. Afro-Asian-Journal-of-Nematology. 1996, 6:1, 40-45.
AB: A new indigenous Steinernema sp. (Rhabditida:Steinernematidae) was recovered from alfalfa [lucerne] fields in the Sultanate of Oman by baiting soil with Galleria mellonella larvae. The thermal niche breadth for establishment (19-37°C), the effect of infective juvenile dosage on establishment, the reproduction in G. mellonella, the LT50 and LC50 to G. mellonella were similar to that of S. riobravis. The new Steinernema was recovered from soil where the indigenous lepidopteran pests were Helicoverpa armigera and Spodoptera littoralis:this new species is likely to be useful for application against the pre-pupae and pupae of the bollworms and leaf worms.

12. Elawad-SA; Gowen-SR; Hague-NGM. 1999. The life cycle of Steinernema abbasi and S. riobrave in Galleria mellonella. Nematology, 1:7-8, 762-764; 14 ref.
AB: S. abbasi completed its development at 30°C between 3.5 and 4.5 days after initial infection, the fastest so far reported for a steinernematid. S. riobrave completed its life cycle after 5 days. Emergence of dauer juveniles (DJ) from infected larvae of Galleria mellonella started earlier when the inoculum was 200 DJ/insect compared to 200 DJ/6 insects.

13. Lee-SangMyeong; Lee-DongWoon; Choo-HoYul; Lee-SM; Lee-DW; Choo-HY. Biology and pathogenicity of the entomopathogenic nematodes Steinernema spp., isolated from forest soil in southern Korea. FRI-Journal-of-Forest-Science-Seoul. 1996, No.53, 117-123.

AB:Laboratory tests were conducted in this study of Steinernema CD-1 and S. GJ-1 entomopathogenic nematodes collected from forest soils in southern Korea Republic. Data are tabulated for mortality rates of Galleria mellonella larvae following application of various concentrations of Steinernema spp. at various temperatures; also for mortality rates of larvae of the Chrysomelid, Agelastica coerulea and the Pyralid, Glyphodes perspectalis [Diaphania perspectalis].