Infection Of An Insect With A Nucleopolyhedrovirus (Lab Report Sample)

Exercise VI: Infection of an Insect with a Bacterium
Telan, Jose Angel Jude B 5/28/18 ENT 275
-2286021844000Abstract
-64770262001000Entomopathogenic bacteria causes lethality and possible mortality when ingested by insects. They enter the gut and could cause various physiological changes and sometimes release toxins detrimental to the health of the insect. One of these bacteria is the Bacillus thuringiensis, or commercially known as Bt. Bt is a crystalliferous bacterium which uses crystals and their resistant spores to cause mortality to their target insects. In this experiment, the students will be familiarized with the infection caused by Bacillus thuringiensis. Two setups were prepared for this experiment, one involved the use of leaf discs while the other involved the use of artificial diet in the setups. Leaf discs were submerged in Bt solution in a petri dish, while Bt suspension were pipetted to small artificial diet cubes in individual cups. Control setups were made which include diet not exposed to Bt. Group of 10 larvae were exposed to the leaf disc setup while individual larvae were placed on each of the cups with artificial diet. The larvae were then collected after 72 hrs. to be dissected and have their tissue streaked on a nutrient agar plate for bacterial isolation. However, second attempt was conducted since the colonies on the first attempt were contaminated with Serratia marcescens. The second attempt resulted in success, thus getting the students familiarized with the Bt infection process.
Introduction
One of the biological entomopathogens include microorganisms like bacteria. They are usually delivered by means of ingestion by the insect, allowing the bacteria to proliferate and develop in the gut, causing lethal symptoms in the insect host. Thus, use of bacteria shows promise in the realm of insect pest management as bacteria could be exploited in order to target certain insect pests, which could reduce economic losses on agricultural farms with these pests. One of the popular commercial bacterium species utilized commercially is the Bacillus thuringiensis or Bt, which is a crystalliferous bacterium (Schnepf et al. 1998). Crystals or parasporal bodies formed by this bacterium, along with the resistant spore that they produce allows them to be effective pathogens against insects. Also, they are amenable to mass production in laboratories thus they are very ideal for biological control studies and application. In this experiment, the students will be allowed to be familiarized with the infection caused by Bacillus thuringiensis on insect larvae.
Materials and Methods
Two general setups could be described in this experiment. One involved the use of leaf discs and one involved the use of artificial diet. In the setups utilizing leaf discs, leaf discs were prepared by cutting into sizes that could fit in petri dishes and disinfecting using 0.05% sodium hypochlorite solution for 5 mins and rinsing in running tap water for 10 minutes before blot drying in paper towels. The leaf discs were then dipped in either sterile distilled water or the Bt suspension before air-drying. One setup represented control where the leaf discs are not exposed to Bt but instead dipped in sterile distilled water, and the other setup used Bt suspension-dipped leaf discs. 10 larvae for each setup were applied on each petri dish and then sealed. On the artificial diet, individual setups were prepared using sealed cups. Cups containing a small 0.5cm x 0.5cm x 05cm of previously prepared artificial diet were overlaid with either 10uL of sterile distilled water or with 10uL of Bt suspension using a micropipette. 10 individual setups were prepared for each, resulting to 20 cups of individual setups. Larvae were placed on each cup after the artificial diet was air-dried for a few minutes. Collectively, the larvae were allowed to feed on the setups and observed for 24, 48, and 72 hours. Basic comparison was made for the larve setups with no Bt exposure as compared to Bt larvae. Once the insect achieves mortality, the cadavers were kept in sterile microcentrifuge tubes for isolation of the entomopathogen.
Results and Discussion
Two attempts were conducted for this experiment. On the first attempt, the prepared petri dishes of the dissected larvae showed colonial growth with obvious red color, which suggests that the colonies growing are Serratia marcescens (Bunting, 1940). This Serratia marcescens is also a bacterium and could also be considered entomopathogenic (Tambong 2013), thus this could suggest that the bacterial species that killed the test specimens are Serratia marcescens or it could also suggest that the colonies were contaminated with Serratia marcescens. The contamination was more likely to have occurred since proper aseptic techniques were not fully implemented thus causing high risk of contamination on the bacterial cultures.
Table 1. Observed mortality on the setups for the second attempt of Bt infection on insect larvae.
Mortality after:

24hrs

48hrs

72hrs

Replicate

1

2

1

2

1

2

Control

5

3

7

6

8

7

Bt-exposed

8

9

10

10

10

10

190503687445Figure 1. One of the petri dish setups showing S. litura larvae cadavers exposed to Bt.0Figure 1. One of the petri dish setups showing S. litura larvae cadavers exposed to Bt.190504889500On the second attempt, the insect larvae achieved mortality within 48 hrs and showed signs of darkenin...