A. Sagel

Volatiles Emitted From Eight Wound-Isolated Bacteria Differentially Attract Gravid Screwworms (Diptera: Calliphoridae) to Oviposit

ABSTRACT Bovine blood inoculated with bacteria isolated from screwworm [Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae) ]-infested animal wounds was tested as an attractant for oviposition for gravid screwworms. Eight species of gram-negative coliform (Enterobacteriaceae) bacteria mixed with bovine blood singly or all species combined and incubated for various times produced volatiles that attracted gravid flies in a cage bioassay in varying numbers. In 15-min duration tests, volatiles from five species of bacteria (Klebsiella oxytoca, Proteus mirabilis, Proteus vulgaris, Providencia rettgeri, and Providencia stuartii) attracted more females than volatiles of the three species (Enterobacter cloacae, Enterobacter sakazakii, and Serratia liquefaciens). In 1-h duration oviposition tests, volatiles from the substrate using the same five species of bacteria attracted more females to oviposit than the other three species. Volatiles from 24-h incubation period elicited least attraction and oviposition whereas volatiles from the 48- and 72-h incubation period resulted in significantly more attraction and oviposition. Attraction and oviposition decreased significantly when the substrates were incubated for 96 h. Volatiles from substrate with all species of bacteria combined attracted a significantly higher percentage of flies to land and oviposit than those from substrates prepared with single species. It is possible that multiple active chemicals present in volatiles of the all-species substrate may act as synergists resulting in greater response than those observed with volatiles from single-species substrate. Before oviposition flies took a bloodmeal from the oviposition substrate. It is possible that the oviposition is moderated by two different factors in screwworm—first, by using a chemical cue to land on a potential oviposition site and second, by using a bloodmeal to stimulate oviposition.

Volatiles from Waste Larval Rearing Media Attract Gravid Screwworm Flies (Diptera: Calliphoridae) to Oviposit

ABSTRACT Gravid screwworm flies, Cochliomyia hominivorax (Coquerel), are attracted to the volatiles from waste larval rearing media to deposit eggs. Studies were conducted to identify volatile chemicals from the waste larval media and determine their effectiveness to attract gravid flies to oviposit. Volatiles were collected using solid-phase microextraction method, and five active chemicals, namely, dimethyl disulfide, dimethyl trisulfide, phenol, p-cresol, and indole, were identified using gas chromatography—mass spectrometry. In electroantennography studies, antennae of gravid screwworm flies, Cochliomyia macellaria (F.), responded positively to each of the identified compounds. A synthetic blend of these five compounds in the ratio of 335:200:57:1:12 was prepared and tested for its effectiveness to attract both C. hominivorax and C. macellaria using laboratory bioassay methods. Significantly more gravid C. macellaria were attracted to and landed on substrates treated with 10-fold diluted blends compared with those landed on substrates treated with ethanol only (as control). Only a few young females and young and old males were attracted to the substrates treated with the synthetic blend. The C. hominivorax females laid significantly more eggs on substrates treated with waste media, 10-fold diluted blend, and 100-fold diluted blend than on substrates with undiluted blend or ethanol. Similarly, C. macellaria deposited significantly more eggs on substrates treated with waste media, 10-fold diluted blend, and 100-fold diluted blend compared with substrates with undiluted blend or ethanol. C. macellaria females deposited significantly less amount of eggs than did C. hominivorax females. These results indicate that the synthetic blend of five compounds identified may serve as an oviposition attractant for C. hominivorax as well as for C. macellaria.

Feasibility of using a Caribbean Screwworm for SIT Campaigns in Brazil

ABSTRACT The screwworm, Cochliomyia hominivorax (Coquerel), remains one of the most damaging parasites of livestock in South America, causing millions of dollars in annual losses to producers. Recently, South American countries demonstrated interest in controlling this pest using the Sterile Insect Technique, and a pilot-project was conducted near the Brazil-Uruguay border in 2009. Since molecular studies have suggested the existence of C. hominivorax regional groups, crossing tests were conducted to evaluate mating competitiveness, mating preference and reproductive compatibility between a C. hominivorax strain from the Caribbean (Jamaica-06) and one from Brazil. Mating rates between Jamaican males and Brazilian females ranged between 82 and 100%, and each male inseminated from 3.3 to 3.95 females. Sterile males, regardless of the strain, competed equally against the fertile males for Brazilian females. Jamaican sterile males and Brazilian fertile males mated randomly with fertile or sterile females. No evidence of genetic incompatibility or hybrid dysgenesis was found in the hybridization crosses. Mating barriers should not compromise the use of Jamaican sterile males for Sterile Insect Technique campaigns in Brazil.

Artificial diets used in mass production of the New World screwworm, Cochliomyia hominivorax

The New World screwworm, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), has been eradicated from North and Central America using the sterile insect technique. This success has been based on mass production of high‐quality screwworms using artificial diets since 1958. Many diet formulations for both larvae and adults have been developed, mainly driven by cost efficiency and material supply. However, only four larval and two adult diet formulations have been applied in the six sterile fly mass production plants in the USA, Mexico and Panama. Herein, we briefly review the history of screwworm diet research and development, introduce the diet formulations used in mass rearing and discuss their advantages and disadvantages in terms of plant application. Finally, we propose future research on screwworm nutrition, potential protein sources, feeding stimulants, further optimization of screwworm formulations and possible methods to reduce the negative qualities of waste generated during the mass production.

The Optimum Diet of Spray-Dried Animal Blood Cells as Protein Source for Adult Screwworms (Diptera: Calliphoridae)

Spray-dried animal blood cells were evaluated as a protein source for mass rearing adult screwworms, Cochliomyia hominiorax Coquerel. Males and females were fed control diets of either a corn syrup...

Evaluation of artificial larval rearing media waste as oviposition attractant for New World screwworms (Diptera: Calliphoridae).

ABSTRACT The waste artificial larval rearing media of New World screwworms, Cochliomyia hominivorax (Coquerel) were evaluated to determine their effectiveness as oviposition attractants. Various concentrations of waste larval media resulting from rearing screwworm larvae in gel and cellulose fiber-based artificial diets tested over a 4-wk period attracted varying number of gravid screwworm flies to oviposit. Three-day-old waste medium with concentrations of 10 and 25% were most attractive to gravid female flies for oviposition and resulted in the most oviposition. One and 7-d-old wastes at all concentrations were less attractive for oviposition than the 3d-old media. The fresh (0-d-old), 14-d- and 28-d-old waste media were the least attractive substrates for oviposition. The waste from the cellulose fiber-based diet resulted in significantly more oviposition compared with waste from the gel-based diet. Microorganisms growing in the waste media probably produce metabolites that attract gravid screwworm flies to oviposit. Use of the waste products of appropriate age and dilution as oviposition substrates would enhance oviposition in mass production colony cages.

Solidifying Agent and Processing of Blood Used for the Larval Diet Affect Screwworm (Diptera: Calliphoridae) Life-History Parameters

ABSTRACT Spray-dried whole bovine blood and a sodium polyacrylate polymer gel as a bulking and solidifying agent are among the constituents of the current larval diet for mass rearing screwworm, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae). Locally available, inexpensive dietary materials could reduce rearing cost and address an uncertain commercial supply of spray-dried blood. We compared efficacy of diet prepared from fresh bovine blood after decoagulation with sodium citrate or ethylenediaminetetraacetic acid (EDTA) or after mechanical defibrination, with the diet containing spray-dried blood using either gel or cellulose fiber as the bulking and solidifying agent. Several life-history parameters were compared among insects reared on each of the blood and bulking agent diets combination. Diets containing citrated blood yielded the lightest larval and pupal weights and fewest pupae. EDTA-treated blood with the gel also caused reductions. EDTA-treated blood with fiber yielded screwworms that were heavier and more numerous than those from the diet with citrated blood but lighter than those from the control diet using spray-dried blood. A reduction in percentage of adults emerging from pupae occurred from diets with both bulking agents using citrated blood and the diet using EDTA mixed with the gel bulking agent. As a group, the cellulose-fiber diets performed better than the gel diets. Larval diet did not affect adult longevity, weight of the eggs deposited by the females that emerged or subsequent egg hatch. Parameter measurements of insects from both defibrinated blood diets were similar to those from the spray-dried blood diets, indicating that fresh, defibrinated bovine blood can successfully replace the dry blood in the screwworm rearing medium.

Distribution and Persistence of Sterile Screwworms (Diptera: Calliphoridae) Released at the Panama–Colombia Border

Abstract The sterile insect technique is used by the Comisión Panamá – Estados Unidos para la Erradicación y Prevención del Gusano Barrenador del Ganado (COPEG) to maintain a barrier at the border of Panama and Colombia to prevent screwworms, Cochliomyia hominivorax (Coquerel), from South America reinfesting North America. Before studying the distribution and persistence of sterilized, mass-produced screwworms released in the barrier zone, the utility of applying fluorescent dust (∼1.0 mg/fly) to pupae and to newly emerged adults was evaluated to determine the potential effect on fly survival. The flight ability of flies collected from two adult emergence/collection systems (enclosed towers and open chambers) and treated with low (∼0.20 mg/fly) or high (∼1.0 mg/fly) amounts of fluorescent powder was compared. The distribution and persistence of sterile screwworms marked with fluorescent powder (∼0.20 mg/fly), after collection from the same two adult emergence/collection systems, was compared after their release in the barrier zone. The results demonstrated that: 1) fluorescent dust did not negatively affect sterile screwworm longevity or flight ability; 2) no differences were detected between sterile flies collected from the two emergence systems; and 3) sterile screwworms distributed evenly in the barrier zone and persisted for > 6 d. This information was useful in implementing the use of a new sterile fly emergence/collection system and deploying a new strain by COPEG for the barrier zone maintenance program; it will be valuable for evaluating alternative release strategies of sterile screwworms by the eradication and barrier maintenance program.

Effects of New Dietary Ingredients Used in Artificial Diet for Screwworm Larvae (Diptera: Calliphoridae)

ABSTRACT Spray-dried whole bovine blood, dry poultry egg, and a dry milk substitute are the constituents of the artificial diet currently used for mass rearing screwworm larvae, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae). Due to high cost and uncertainty of the commercial supply of spray-dried blood, research was conducted to identify alternative, locally available, inexpensive, dietary ingredients which could reduce cost of rearing and eliminate concerns of short supply. Experimental diets were prepared without blood component and with various ratios of bovine blood or blood cell product and defatted soy flour. Results indicate that spray-dried bovine blood can be replaced by a readily available and less expensive blood cell product. When the quantity of whole dried blood or blood cell component was reduced or removed completely from the diet, the larvae did not feed adequately, resulting in high mortality. Those larvae that survived produced pupae that were of unacceptable quality. When the milk product was replaced by soy flour, pupae were slightly smaller than those reared using the current diet; however, replacement of egg product with soy flour produced even smaller pupae. Longevity of adult flies that emerged from these small pupae was short and the females deposited few eggs. These results indicate that soy flour cannot replace the blood component from the diet, but can replace the milk product successfully. It is likely that some factor or a combination of factors in the blood act as feeding stimulants, without which larvae are unable to feed normally, resulting in high larval mortality.

Semiochemicals released from five bacteria identified from animal wounds infested by primary screwworms and their effects on fly behavioral activity

Background The Primary screwworm, Cochliomyia hominivorax (Coquerel), is a serious pest feeding on living flesh of any warm-blooded animal, including humans. It was eradicated from the United States in the early 1980s using the sterile male technique. However, it was recently detected in populations of wild deer and pets in the Florida Keys of the US. For monitoring purposes, screwworm flies are normally trapped using attractant bait with liver. However, there has been little effort to develop an efficient monitoring system for detection of screwworm flies using a specific synthetic attractant blend. Several studies have shown that odors from animal wound fluids attract screwworm adults, particularly gravid females. Bacteria associated with animal wounds have been identified that act as a major source for this attraction. To understand what volatiles attract screwworms we inoculated bovine blood with previously identified bacteria. We identified volatile chemicals released from the inoculated blood and other selected media over time and assessed the effect of those chemicals on behavioral activity of adult screwworm flies. Methodology/Principal findings A total of 7 volatile compounds were collected from bacteria incubated in either broth or blood using solid-phase microextraction, and their chemical structures were identified by their characteristic mass spectrum fragments and confirmed by retention times in comparison to those of synthetic standards via gas chromatograph combined mass spectrometry analyses. Five major volatiles including dimethyl disulfide, dimethyl trisulfide, phenol, p-cresol and indole were detected from a mixture of 5 bacteria incubated in blood. The ratios of volatiles released differed among different incubation media, time and individual bacteria. A synthetic mixture containing the five compounds was demonstrated to be attractive to adult screwworm flies both in laboratory assays and field trapping trials. Conclusions/Significance The results obtained from this study may assist in developing an efficient trapping system using the identified attractant blend to detect the infestation of primary screwworms. This is also the first study to explore the complex systems in volatile release profiles from 5 bacteria isolated from screwworm-infested animal wounds that are incubated with different media and incubation time, as well as individual and multi-species bacterial communities.