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Entomopathogenic Nematode Sampling In Soil From A Termite Habitat Located In University Of The Philippines Los Banos (Research Paper Sample)


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TELAN, Jose Angel Jude B.
Special Project
Submitted to Dr. Barbara L. Caoili
in Partial Fulfillment to
ENT 275: Insect Pathology
-17145258892900Entomopathogenic nematodes are nematodes that are able to cause detrimental changes in insects that they infect. They are generally composed by Heterorhabditis spp. and Steinernema spp. Symbiotic bacteria that the infective juvenile stage harbor helps in causing rapid mortality to the potential host insect. This characteristic, along with amenability to mass production, availability of use with some insecticides, easy application, and safety to nontarget organisms allow these EPNs to have a good potential as a biological control agent that could be applied in many insect pest management strategies. As they are naturally soil-inhabiting, these EPNs could be usually found on subterranean insects such as termites. In this special project, soil sample will be conducted in order to collect and identify the nematode that was observed during the dissection of the termite gut in one of the laboratory exercises of the ENT 275 class under Dr. Barbara Caoili. Baiting setups using the soil collected from the vicinity of the collected termite habitat was prepared. White traps were made for the insect cadavers collected from the baiting setups in order to observe the emerging IJs. IJs were collected and observed under the microscope but was identified as non-members of the families Steinernematidae and Heterorhabditidae thus revealing that the nematode is not an EPN.
Nematodes are members of the Phylum Nematoda and could be described as one of the most abundant animals on Earth. They are commonly known as parasites of other organisms but may also possess free-living forms in various environments such as soil and bodies of water. Among invertebrates, one of the living things that they parasitize on are insects. Insects could reach mortality once infected by these nematodes labeled as entomopathogenic nematodes or EPNs. Popular nematode families that are studied for application in insect biological control are Steinernematidae and Heterorhabdidtidae. These nematode groups are mutualistically associated with bacteria Xenorhabdus and Photorhabdus. (Kaya & Gaugler 1993) Lethality of these nematodes are mainly attributed to the bacteria that they are associated with. The infection process first involved the host searching of the infective juveniles or IJs. Afterwards, the IJs develop and proliferate inside the host, releasing the bacteria inside. Once the insect host dies, the progeny of the now-adult nematodes develop into IJS, depleting the nutrients of the insect cadaver. Once the nutrients deplete, the IJs emerge from the empty body and will conduct host searching once again.
Nematode behavior towards host-searching involved their attraction to physical and chemical cues leading to their prey. There is a recent study which suggests that nematodes are directionally stimulated towards electromagnetic stimuli (Shapiro-Ilan et al. 2012). Some nematodes could prefer to be ambushers or cruisers in terms of host-searching behavior. Ambushers like Steinernema carpocapsae are ideally energy-savers as they remain sedentary while waiting for prey to be “ambushed”. Ideal prey for ambushers include highly mobile insects such as mole crickets. Cruisers such as Steinernema glaseri highly respond to insect host cues and will be actively pursuing their target since they are highly mobile. Ideal targets for these cruisers would be mostly sedentary insects found in the soil. (Lewis et al. 1992, 1993) Next behavior would be the host-attachment, which is the pre-requisite for infection. Some nematodes, especially ambushers, exhibit the so-called “nictating” behavior which could be described as the lifting of the nematode body aside from the posterior portion, attracting potential insect hosts. (Campbell et al. 1993, Grewal et al. 1994) Host recognition and specificity relies on the surface carbohydrates present on their target insect which is detected by the nematode (Zuckerman and Jansson 1984). Lastly, the penetration of the nematode on the potential insect host. Penetration should be relatively easy for Heterorhabditids since they possess a ‘tooth’ which allows them to penetrate intersegmental tissues to enter not just the orifices (Bedding & Molyneux 1982). Unlike Steinernematids which rely on the openings in order to penetrate the insect and proliferate inside. They are able to persist in the environment due to their hardy nature, but they could easily be affected by desiccation and enough heat (Kung et al. 1991). Understanding of their behavior allows one to gain understanding on their usual cycles and exploit it for applications in other fields like biological control.
Promising development of studies concerning these nematodes as application for biological control since they have broad host range (Kaya & Gaugler 1993), safe to nontarget organisms, does not need special equipment to be applied (Jansson & Lecrone 1994), amenable to genetic selection, and could be applied with some chemical pesticides (Forschler et al. 1990). However, as with some insect biological control agents, these EPNs could be applied better if they are highly focused on their target insect with less risk of targeting other organisms to reduce mortality of nontarget organisms. Thus, EPNs that are collected in insects from the wild could be valuable information as it could lead to a record of an EPN which preferred certain insects over others. The coincidence of an accidental discovery in a common laboratory exercise is not high. But in one of the exercises conducted for the subject entitled Insect Pathology (ENT 275), we have found a nematode in one of the dissected termites and was preliminarily identified as an EPN. In this study, collection of the said nematode will be conducted, and morphological analysis will be performed to confirm the identity of the said nematode. Specifically, the study aims to:
1 To sample the area where the termite was previously collected,
2 To collect nematodes from the soil sample,
3 To confirm if the collected nematode belongs to the entomopathogenic nematode families.
293456649616600Materials and Methods
-6352167255Figure 1a & 1b. Soil collection area featuring the termite habitat.0Figure 1a & 1b. Soil collection area featuring the termite habitat.Soil collected on one of the termite habitats from the Insectary Area located at the back of UPLB Biological Sciences building were sampled for nematodes using insect baiting (Figure 1). Insect baiting method is where living insect larvae are subjected to closed containers with soil samples. After subjecting the living larvae that served as baits, the setups are then observed for mortalities which are then collected and isolated in a microassay plate. The materials and equipment used were provided by the Insect Pathology Laboratory under Dr. Barbara Caoili. The collected nematodes in microassay plates are then observed for symptoms of EPN infection such as discoloration and emerging nematodes. Once the infective juveniles or IJs emerge, the cadaver is now then transferred aseptically into setups we call as “white traps”. White traps are sealed petri dishes with enough water just to fill the surface where a microplate lined with a moist filter paper holding the insect cadaver. Since IJs -263467170081900prefer bodies of water after emerging, the water 184259761058200-1306375311601Figure 2. From top to bottom. Top left: White trap containing one of the lesser waxmoth larvae isolated. Top right: microassay plates used for individual cadavers. Bottom: Containers used to store water with IJs collected from white traps.Figure 2. From top to bottom. Top left: White trap containing one of the lesser waxmoth larvae isolated. Top right: microassay plates used for individual cadavers. Bottom: Containers used to store water with IJs collected from white traps.-27813064273600will hold the IJs in the white trap (Glazer & Lewis 2000). Once the water in the setup could be observed for IJs, it should be collected for storage and identification of the nematodes. Afterwards, these IJs could be used for reinfection of insect larvae in order to confirm their entomopathogenic characteristic. Once the reinfected larvae were subjected to white trap again, the IJs collected will be identified again and hopefully should be the same species with the previously collected nematode for confirmation that it is an EPN.
Results and Discussion
262655672512Figure 4. Photos of the second batch of nematodes collected from the samples. 0Figure 4. Photos of the second batch of nematodes collected from the samples. 2935605347529700279403468947003457990608800292175190597200348103204845Figure 3. Photos of the first batch of nematodes collected from the samples featuring the anterior part of the nematodes. 0Figure 3. Photos of the first batch of nematodes collected from the samples featuring the anterior part of the nematodes. First isolation of the setups was conducted for EPN identification in the laboratory. The isolated EPNs were identified as “non-EPN” since there is an absence of the bacterial pouch which is present on Steinernematids and the presence of the barrel-shaped stoma and obvious tripartite esophagus which are indicative character of the Rhabditids (Shah & Mahamood 2017) (Figure 3). This result could imply the following: the concentration of EPN is in very low titer that was not able to compete with the volume of non-entomopathogenic nematodes, the setups were contaminated with non-entomopathogenic nematodes after ...
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