Attachment_1,_Protocols.pdf

APPENDIX A - PROTOCOLS FOR THE EVALUATION OF MOLDS, YEASTS AND

BACTERIA IN SOLID DIET INGREDIENTS INCLUDUING CORN COB SAMPLES

Prepared by: Sandra Leal (PPQ Harlingen); Don Thomas (ARS); Bacilio Salas, Don Vacek, and Hugh Conway (PPQ CPHST Mission Lab); Kristy Gottfried PPQ Mexican Fruit Fly Mass Rearing Facility

Use all sterile equipment, under aseptic and antiseptic techniques.

Five random samples will be obtained from each shipment (five different bags)

There are several microbes of concern for the mass rearing of the Mexican fruit fly (Anastrepha ludens). Among the molds, Aspergillus niger, Aspergillus flavus, Fusarium sp. are the most important for contamination as well as for their toxigenic effect to rearing insects. Among bacteria, Morganella morganii, Morganella psychrotolerans, Morganella sp., Providencia rettgeri and Providencia alcalifaciens can seriously reduce the yield of larvae (50-100%) during Mexican fruit fly production, and the yeast Zygosaccharomyces bailii can reduce larval yield (25-35%.

Isolation of molds and yeast (standard microbial media testing)

Prepare culture plates of Difco™ Sabouraud Dextrose Agar for the isolation of Aspergillus spp. and other molds, Malt Yeast Agar for the isolation of yeasts, and Komada’s medium for the isolation of Fusarium spp.

Sift 10 mg of material to test (ie. corn cob) on each culture plate. Incubate culture plates at 28oC, and transfer distinct morphotype of mold or yeast onto new culture plates as soon as they are visible for further identification. Note: Except on Komada’s medium, slow growing mold colonies in culture plates are readily overgrown by Rhizopus spp.

Isolation of bacteria (standard microbial media testing)

Prepare culture plates of Difco™ XLD Agar and/or Difco™ Pseudomonas Agar F amended with Bravo WS (10 ppm)for the isolation of bacterial species.

Sift directly 10 mg or perform serial dilutions of corn cob and plate on culture plates, or place 1g corn cob in 99 ml sterile water, incubate for 24 h at 28oC, then perform dilutions series. Incubate culture plates at 28oC for 48 hrs. Transfer unique morphotype of different colonies onto new culture plates. Purify bacterial isolates and send for identification

Test suspicious bacterial isolates for their pathogenicity to Mexican fruit fly using small cup bioassays.

Problematic bacteria, yeasts, and fungi

APPENDIX B - PROTOCOL FOR THE EVALUATION OF NEMATODES AND

PARASITES IN SOLID DIET INGREDIENT INCLUDING CORN COB SAMPLES

Prepared by: Sandra Leal (PPQ Harlingen); Don Thomas (ARS); Bacilio Salas, Don Vacek, and Hugh Conway (PPQ CPHST Mission Lab); Kristy Gottfried PPQ Mexican Fruit Fly Mass Rearing Facility

Identification of harmful nematodes and parasitic roundworms

The following nematodes and parasitic roundworms have been identified thus far:

Parasitic worm – found to infect all life stages of the Mexican fruit fly.

Procephalobus sp. Panagrolaimidae – egg laying panels and all life stages

Procephalobus mycophilus Panagrolaimidae – egg laying panel and all life stages

Mononchus sp. Mononchidae – found infesting egg laying panels

For Real-Time evaluation of adult forms of nematodes and parasites

• Use all sterile equipment, under aseptic and antiseptic techniques.

• Reconstitute 50 g of dry corn cob or diet ingredient sample with 50ml of sterile NaCl

• Prepare 10 fresh smears, on a 3” X 1” microscope slide and proceed to stain 5 slides with Hematoxylin and Eosin, mount and apply cytology fixative to all of the slides.

These stained and unstained slides will confirm the presence or absence of adult forms of nematodes and parasites while being evaluated under Phase microscopy and/or Epifluorescence.

For evaluation of immature trophozoites and sporozoites forms of nematodes and parasites

• Inoculate 10 sterile, Tryptone-based 100mm Nematode Growth Media plates and incubate aerobically at 32C.

Microscopic observation and microphotograph/ video documentation is required at 6 hours intervals for the first 12 hours from inoculation, at 24 hours, and subsequently at every 12 hours intervals until 96 hours have been completed.

Where to look for harmful microbial contaminants

It is already known that live parasites reside in the foamies and have very likely contaminated other rearing processes.

Observation of parasitic worm in different life stages of Mexican fruit fly

APPENDIX B - PROTOCOL FOR THE EVALUATION OF NEMATODES AND

PARASITES IN SOLID DIET INGREDIENT INCLUDING CORN COB SAMPLES

Prepared by: Sandra Leal (PPQ Harlingen); Don Thomas (ARS); Bacilio Salas, Don Vacek, and Hugh Conway (PPQ CPHST Mission Lab); Kristy Gottfried PPQ Mexican Fruit Fly Mass Rearing Facility

Identification of harmful nematodes and parasitic roundworms

For Real-Time evaluation of adult forms of nematodes and parasites

• Use all sterile equipment, under aseptic and antiseptic techniques.

• Reconstitute 50 g of dry corn cob or diet ingredient sample with 50ml of sterile NaCl

• Prepare 10 fresh smears, on a 3” X 1” microscope slide and proceed to stain 5 slides with Hematoxylin and Eosin, mount and apply cytology fixative to all of the slides.

These stained and unstained slides will confirm the presence or absence of adult forms of nematodes and parasites while being evaluated under Phase microscopy and/or Epifluorescence.

For evaluation of immature trophozoites and sporozoites forms of nematodes and parasites

• Inoculate 10 sterile, Tryptone-based 100mm Nematode Growth Media plates and incubate aerobically at 32C.

Microscopic observation and microphotograph/ video documentation is required at 6 hours intervals for the first 12 hours from inoculation, at 24 hours, and subsequently at every 12 hours intervals until 96 hours have been completed.

Where to look for harmful microbial contaminants

It already is known that live parasites reside in the foamies and have very likely contaminated other rearing processes.

Determination of Effect of culture media and incubation time and temperature on growth and development of nematodes and possible symbiotic bacteria

Inoculations were performed under Aseptic and Antiseptic techniques as follows:

White (sterile) area incubated at 15-20C under Aerobic (95% air 5% CO2) and Anaerobic conditions (Anaerobic Chamber).

Grey area was kept at room temperature (25C) under Aerobic conditions.

The specimens were inoculated using the following media:

Nutrient Media (non-selective)

Maltose-based culture media

Eosin-Methylene blue selective medium

Differential medium including Natural Red and Eosin

Beta-Lactamase markers for Gram stained bacteria

The USDA requires corn cob grits and all diet ingredients to be free of microbial contamination for mass rearing of Mexican fruit flies. CPHST recommends that a source of corn cob grits free of microbial contamination be found, purchased, and used for Mexican fruit fly mass rearing.