A molecular approach to identifying the natural prey of the African creeping water bug Naucoris, a potential reservoir of Mycobacterium ulcerans.

The extra-oral digestion of creeping water bugs (Naucoridae: Hemiptera) hinders the study of their diet using the standard method of identifying prey body parts in the gut. Genetic methods are available, but rely on PCR tests or similar diagnostics to confirm suspected prey. Where the potential prey is unknown and a broad search for all possible prey is desirable, methods that can potentially capture any prey item are required. Naucoris sp. is known to harbor Mycobacterium ulcerans (Actinomycetales: Mycobacteriaceae), the causative bacterium of Buruli ulcer. Outbreaks of Buruli ulcer have been associated with disturbed freshwater habitats, but the mode of transmission to humans remains unclear. Here we examine the diet of Naucoris sp., a dominant aquatic predator in water bodies in Ghana where the prevalence of Buruli ulcer is high. We cloned and sequenced 576 PCR products (mtDNA rrnL, cox1) isolated from the gut of 60 Naucoris sp. individuals to determining diet composition as broadly as possible. Using phylogenetic analysis of newly sequenced clones and 6 potential prey taxa collected from the site, sequences isolated from Naucoris sp. guts matched locally collected Coleoptera (Hydrophilidae). Blastn queries to GenBank of other clone sequences produced matches to (Anura) (n = 1), Rotifera (n = 5), and fungi (n = 4) as additional components of the diet. Our results suggest that sp. in this Buruli ulcer-endemic area feeds on a wide range of prey and body sizes, and that the approach could be successfully applied to studies of aquatic food webs where morphological identification of prey is impossible and where little or no a priori knowledge is available.

Interaction of Mycobacterium ulcerans with mosquito species: implications for transmission and trophic relationships.

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a severe necrotizing skin disease that causes significant morbidity in Africa and Australia. Person-to-person transmission of Buruli ulcer is rare. Throughout Africa and Australia infection is associated with residence near slow-moving or stagnant water bodies. Although M. ulcerans DNA has been detected in over 30 taxa of invertebrates, fish, water filtrate, and plant materials and one environmental isolate cultured from a water strider (Gerridae), the invertebrate taxa identified are not adapted to feed on humans, and the mode of transmission for Buruli ulcer remains an enigma. Recent epidemiological reports from Australia describing the presence of M. ulcerans DNA in adult mosquitoes have led to the hypothesis that mosquitoes play an important role in the transmission of M. ulcerans. In this study we have investigated the potential of mosquitoes to serve as biological or mechanical vectors or as environmental reservoirs for M. ulcerans. Here we show that Aedes aegypti, A. albopictus, Ochlerotatus triseriatus, and Culex restuans larvae readily ingest wild-type M. ulcerans, isogenic toxin-negative mutants, and Mycobacterium marinum isolates and remain infected throughout larval development. However, the infections are not carried over into the pupae or adult mosquitoes, suggesting an unlikely role for mosquitoes as biological vectors. By following M. ulcerans through a food chain consisting of primary (mosquito larvae), secondary (predatory mosquito larva from Toxorhynchites rutilus septentrionalis), and tertiary (Belostoma species) consumers, we have shown that M. ulcerans can be productively maintained in an aquatic food web.

Examination of nocturnal blow fly (Diptera: Calliphoridae) oviposition on pig carcasses in mid-Michigan.

The most common application of forensic entomology involves estimating a portion of the postmortem interval (PMI), which usually assumes that blow flies (Diptera: Calliphoridae) do not oviposit nocturnally. Research objectives were to (1) investigate blow fly nocturnal oviposition in relation to sunrise and sunset in Michigan; (2) evaluate abiotic variables postulated to affect blow fly oviposition; and (3) conduct laboratory experiments testing blow fly activity under complete darkness. In 2006, nocturnal oviposition was evaluated in relation to sunset by exposing pigs to fly colonization at 1-h intervals, beginning 2 h before and ending 2 h after sunset. This test was replicated in 2007; however, replicate pigs were placed in the field 2 h after sunset, and hourly observations were made into the following morning. Oviposition was never observed at night. In a laboratory experiment, Lucilia sericata (Meigen), never oviposited on liver hanging above or placed directly on the ground in a completely dark room, Another dark room laboratory study documented that adult flies launched into the air could not fly. This study documents that the probability of nocturnal oviposition on pig carcasses in Michigan was extremely low to nonexistent. These results should be considered when estimating a portion of the PMI in forensic entomological investigations.

Aquatic invertebrates as unlikely vectors of Buruli ulcer disease.

Buruli ulcer is a necrotizing skin disease caused by Mycobacterium ulcerans and associated with exposure to aquatic habitats. To assess possible transmission of M. ulcerans by aquatic biting insects, we conducted a field examination of biting water bugs (Hemiptera: Naucoridae, Belostomatidae, Nepidae) in 15 disease-endemic and 12 non-disease-endemic areas of Ghana, Africa. From collections of 22,832 invertebrates, we compared composition, abundance, and associated M. ulcerans positivity among sites. Biting hemipterans were rare and represented a small percentage (usually <2%) of invertebrate communities. No significant differences were found in hemipteran abundance or pathogen positivity between disease-endemic and non-disease-endemic sites, and between abundance of biting hemipterans and M. ulcerans positivity. Therefore, although infection through insect bites is possible, little field evidence supports the assumption that biting hemipterans are primary vectors of M. ulcerans.

Persistent association of Mycobacterium ulcerans with West African predaceous insects of the family belostomatidae.

A number of studies have suggested that Mycobacterium ulcerans, the etiological agent of Buruli ulcer, may be transmitted to humans by insect bites. M. ulcerans has been isolated from a predaceous aquatic insect, and PCR detection of M. ulcerans DNA in aquatic environments suggests that the organism is widely distributed within many invertebrate taxa and functional feeding groups. Thus, M. ulcerans may be concentrated through different trophic links. However, the specific environmental niche of M. ulcerans and route of transmission to humans remain a mystery. In this study, a biologically relevant infection model in which M. ulcerans-infected mosquito larvae were fed to a species of predaceous hemiptera (African Belostomatidae) was used to demonstrate the persistent colonization of M. ulcerans and subsequent transmission of bacteria to naïve prey. The association of M. ulcerans with specific anatomical compartments showed that M. ulcerans accumulates preferentially on the exoskeleton. In contrast, few organisms were found in dissected guts or salivary glands. No difference was found between the ability of wild-type M. ulcerans and an M. ulcerans isogenic mycolactone-negative mutant to colonize belostomatids. These data show that African belostomatids can successfully be colonized by M. ulcerans and support the trophic transfer of M. ulcerans within the environment.

Caddisflies assist with homicide case: determining a postmortem submersion interval using aquatic insects.

Although few indicators of time since death for corpses found in aquatic ecosystems are comparable in precision to the insect indicators used in terrestrial cases, there are observations that can be useful in suggesting or ruling out an approximate PMSI (postmortem submersion interval). For example, the time intervals required for certain growth phases of aquatic insects, such as caddisflies, that may attach themselves to the submerged remains can be used to estimate a minimum PMSI. Approximately 8 of the 13 orders of insects containing species with aquatic or semi-aquatic stages are likely to be associated with carrion or corpses in aquatic habitats. We present a case study in which portions of a body from an adult male were discovered in a south central Michigan stream. The body was dismembered and portions were recovered from two bags floating and submerged in the stream. Insect specimens collected from mesh and plastic bags consisted of one fly larva belonging to the family Muscidae, and caddisfly larvae belonging to two families: the Limnephilidae. (case-makers) and the Hydropsychidae, (net spinners). We used unique case-building behaviors of the limnephilid caddisflies found on the remains to elucidate a PMSI range consistent with the disappearance of the victim. It is important for forensic investigators to understand that although some precision is lost in estimating a PMSI with aquatic insects, these organisms should not be ignored in gathering evidence from aquatic crime scenes, and in fact, they can provide valuable details in estimating a PMSI.

Cutaneous myiasis arising in an eccrine adnexal neoplasm.

Myiasis is the infestation of human tissue by fly maggots. Although it is most often reported in tropical and sub-tropical regions of the world and in travelers returning from these areas, cases do occur in the United States. We report here a case of cutaneous myiasis observed in the setting of an eccrine adnexal neoplasm in an otherwise healthy host. Entomological analysis of the isolated organisms revealed additional information of interest.

Collembola of the grave: a cold case history involving arthropods 28 years after death.

This report describes a cold case in which a cadaver of a 28-year-old female was exhumed in February 2005 from a cemetery in Battle Creek, Michigan. She had sustained a gunshot wound to the head and was found dead in her home on November 15, 1977. The body of the victim was subsequently embalmed and then buried at a depth of 1.8 m in an unsealed casket that was placed inside an unsealed cement vault. The exhumation yielded thousands of live specimens of a single species of the order Collembola or spring tails, Sinella (Coecobrya) tenebricosa (Entomobryidae). This species is considered to be a "tramp" species, cosmopolitan in the United States and Canada. Due to the ideal environmental conditions at the site, the population of this species underwent growth and development inside the casket for a number of years. Collected with the Collembola were large numbers of Acarina (mites) of the Family Glycyphagidae, and fly puparia, Conicera tibialis Schmitz (Order: Diptera, Family: Phoridae), also known as coffin flies. These invertebrates are sometimes mentioned by forensic investigators as occurring on corpses in graves, but aspects of their life history are rarely described. The species of Collembola that was found surviving and reproducing on this corpse in a casket exhumed after 28 years was the oldest reported grave site occurrence for any collembolan species based on a survey of the literature back to 1898.

Functional Diversity as a New Framework for Understanding the Ecology of an Emerging Generalist Pathogen.

Emerging infectious disease outbreaks are increasingly suspected to be a consequence of human pressures exerted on natural ecosystems. Previously, host taxonomic communities have been used as indicators of infectious disease emergence, and the loss of their diversity has been implicated as a driver of increased presence. The mechanistic details in how such pathogen-host systems function, however, may not always be explained by taxonomic variation or loss. Here we used machine learning and methods based on Gower's dissimilarity to quantify metrics of invertebrate functional diversity, in addition to functional groups and their taxonomic diversity at sites endemic and non-endemic for the model generalist pathogen Mycobacterium ulcerans, the causative agent of Buruli ulcer. Changes in these metrics allowed the rapid categorisation of the ecological niche of the mycobacterium's hosts and the ability to relate specific host traits to its presence in aquatic ecosystems. We found that taxonomic diversity of hosts and overall functional diversity loss and evenness had no bearing on the mycobacterium's presence, or whether the site was in an endemic area. These findings, however, provide strong evidence that generalist environmentally persistent bacteria such as M. ulcerans can be associated with specific functional traits rather than taxonomic groups of organisms, increasing our understanding of emerging disease ecology and origin.

Lack of effects of Bacillus sphaericus (Vectolex) on nontarget organisms in a mosquito-control program in southeastern Wisconsin: a 3-year study.

A 3-year study (2000-2002) in southeastern Wisconsin was conducted to assess the effects of Bacillus sphaericus applied for mosquito control on nontarget wetland invertebrates. The experimental design consisted of control and treatment sites (that were applied by helicopter with Vectolex CG), each in 2 vegetation habitat types: reed canary grass marsh (Phalaris arundinacea) and cattail marsh (Typha spp.). In each of these areas, a predetermined number of timed (30-sec) D-frame aquatic net samples containing vegetation, detritus, and invertebrates were collected 1 day before spraying and 72 h after spraying to detect for effects. We examined and compared 5 bioassessment measures to determine if there was an effect of B. sphaericus on nontarget organisms during each of the sampling years. The metrics tested were (1) mean taxa richness (the mean number of all taxa), (2) mean diversity (combines taxa richness and abundances in a summary statistic; i.e., Shannon Index [H'I]), (3) Diptera richness (minus mosquitoes) as a proportion of all other taxa richness (Diptera/others richness), (4) Diptera abundance (minus mosquitoes) as a proportion of all other invertebrate abundance (Diptera/others abundance), and (5) functional group changes in percent collector-gatherers, collector-filterers, scrapers, shredders, and predators. When Vectolex was applied during 6 treatments at the labeled dosage rate in the above habitats in Brookfield, WI, no detrimental effects to nontarget organisms could be attributed to this microbial insecticide. Much of the variation in the control vs. treatment and pre vs. post plots was attributed to factors other than the effects of B. sphaericus on nontarget organisms, such as the time of sampling, natural variation that occurs in such diverse habitats as canary grass and cattail marshes, and water depth, which varied among years.

Diel feeding periodicity of larval anopheline mosquitoes on microorganisms and microinvertebrates: a spatial and temporal comparison of Anopheles quadrimaculatus (Diptera: Culicidae) diets in a Michigan pond.

Diel feeding activity of third and fourth instars of Anopheles quadrimaculatus Say were studied in a Michigan permanent pond. This field study examined the consumption of microbial and microinvertebrate food resources over a diel (24-h) period between two habitats (open water and vegetated areas). A fluorochromatic stain (4,6-diamidino-2-phenylindole) was used to quantify microbial dietary components within larval guts and habitats. Microbial analyses show that bacteria were the most abundant food type, followed by detritus, algae, and invertebrate parts/protozoans (IPP). Larval consumption of cladocerans was significantly greater at midnight than noon. Larval gut analyses examined every hour over a 24-h period provided significant evidence as to the approximate time larvae switch microinvertebrate dietary resources. Habitat had a significant effect on microinvertebrate consumption by An. quadrimaculatus larvae. Larvae consumed more water mites and rotifers in the open water areas than vegetated zones. We found that An. quadrimaculatus larvae do not preferentially feed on microinvertebrates over a diel period, however, larvae may feed selectively on rotifers in open water habitats. Knowledge of the "feeding area" or microhabitats, with respect to where and when larvae optimally forage as well as particle sizes and food types consumed in the natural habitats will enhance the success of bacteria and other particulate larvicides.

The effect of an industrial effluent on an urban stream benthic community: water quality vs habitat quality.

We studied the effect of an industrial effluent on the water quality, habitat quality, and benthic macroinvertebrates of an urban stream in southwestern Michigan (USA). The effluent affected water quality by raising in-stream temperatures 13-18 degree C during colder months and carrying high amounts of iron (> 20 x higher than ambient) that covered the streambed. The effluent also affected habitat conditions by increasing total stream discharge by 50-150%, causing a significant change in substrate and flow conditions. We used three methods to collect benthic macroinvertebrates in depositional and erosional habitats and to understand the relative importance of habitat quality and water quality alterations. Macroinvertebrate response variables included taxonomic richness, abundance, and proportional abundance of sensitive taxonomic groups. Results indicated that the effluent had a positive effect on macroinvertebrate communities by increasing the quantity of riffle habitat, but a negative effect on macroinvertebrate communities by reducing water quality. Results illustrated the need for careful consideration of habitat quality and water quality in restoration or remediation programs.

Pig decomposition in lotic aquatic systems: the potential use of algal growth in establishing a postmortem submersion interval (PMSI).

While algal community composition has been examined as a qualitative indicator of postmortem submersion interval (PMSI), there have been no quantitative studies on using algal growth rates as PMSI estimators. The present study was undertaken to examine pig decomposition in streams and to develop a more quantitative approach to estimate a PMSI. Pigs and ceramic tiles were completely submerged and regularly sampled for periphyton growth. Five stages of decomposition were identified for the submerged pig carcasses according to physical characteristics. Algal growth rates, measured quantitatively as a function of chlorophyll-a concentration, were greater on pigs compared with tiles; however, microhabitat (pools versus riffles) did not significantly influence algal growth. Additionally, there was a strong correlation between algal growth rate and time on pigs and tile substrates. This strong correlation was observed after significant rain events. Our study documents for the first time a quantitative technique to determine the length of time a corpse has been submerged in water. We suggest that algal growth rates may be a useful quantitative indicator in criminal investigations involving corpses that are completely submerged in stream or riverine habitats.

Mycobacterium ulcerans fails to infect through skin abrasions in a guinea pig infection model: implications for transmission.

Transmission of M. ulcerans, the etiological agent of Buruli ulcer, from the environment to humans remains an enigma despite decades of research. Major transmission hypotheses propose 1) that M. ulcerans is acquired through an insect bite or 2) that bacteria enter an existing wound through exposure to a contaminated environment. In studies reported here, a guinea pig infection model was developed to determine whether Buruli ulcer could be produced through passive inoculation of M. ulcerans onto a superficial abrasion. The choice of an abrasion model was based on the fact that most bacterial pathogens infecting the skin are able to infect an open lesion, and that abrasions are extremely common in children. Our studies show that after a 90d infection period, an ulcer was present at intra-dermal injection sites of all seven animals infected, whereas topical application of M. ulcerans failed to establish an infection. Mycobacterium ulcerans was cultured from all injection sites whereas infected abrasion sites healed and were culture negative. A 14d experiment was conducted to determine how long organisms persisted after inoculation. Mycobacterium ulcerans was isolated from abrasions at one hour and 24 hours post infection, but cultures from later time points were negative. Abrasion sites were qPCR positive up to seven days post infection, but negative at later timepoints. In contrast, M. ulcerans DNA was detected at intra-dermal injection sites throughout the study. M. ulcerans was cultured from injection sites at each time point. These results suggest that injection of M. ulcerans into the skin greatly facilitates infection and lends support for the role of an invertebrate vector or other route of entry such as a puncture wound or deep laceration where bacteria would be contained within the lesion. Infection through passive inoculation into an existing abrasion appears a less likely route of entry.

Aquatic macroinvertebrate assemblages of Ghana, West Africa: understanding the ecology of a neglected tropical disease.

Buruli ulcer (BU) is an emerging, but neglected tropical disease, where there has been a reported association with disturbed aquatic habitats and proposed aquatic macroinvertebrate vectors such as biting Hemiptera. An initial step in understanding the potential role of macroinvertebrates in the ecology of BU is to better understand the entire community, not just one or two taxa, in relation to the pathogen, Mycobacterium ulcerans, at a large spatial scale. For the first time at a country-wide scale this research documents that M. ulcerans was frequently detected from environmental samples taken from BU endemic regions, but was not present in 30 waterbodies of a non-endemic region. There were significant differences in macroinvertebrate community structure and identified potential indicator taxa in relation to pathogen presence. These results suggest that specific macroinvertebrate taxa or functional metrics may potentially be used as aquatic biological indicators of M. ulcerans. Developing ecological indicators of this pathogen is a first step for understanding the disease ecology of BU and should assist future studies of transmission.

Fish and amphibians as potential reservoirs of Mycobacterium ulcerans, the causative agent of Buruli ulcer disease.

BACKGROUND: Buruli ulcer is a skin disease often associated with proximity to certain water bodies in Africa. Much remains unknown about the reservoir and transmission of this disease. Previous studies have suggested that fish may concentrate Mycobacterium ulcerans, the etiological agent of the disease, in their gills and intestines and serve as passive reservoirs of the bacterium. The objective of this study was to test the hypothesis that fish and amphibians serve as natural reservoirs of M. ulcerans or other closely related mycolactone-producing mycobacteria. METHODS: Polymerase chain reaction targeting the enoyl reductase (ER) domain present in mlsA, which is required for mycolactone production, was used to screen water, fish, and amphibians from water bodies in Ghana for the presence of mycolactone-producing mycobacteria, and positive specimens were subjected to variable number tandem repeat (VNTR) typing. RESULTS: The use of VNTR typing revealed the presence of Mycobacterium liflandii in a tadpole and a fish, and M. ulcerans in an adult frog. Similarity percentage analysis (SIMPER) showed that the predatory cichlid Hemichromis bimaculatus was associated with ER-positive water bodies. No amphibian species or fish-feeding guild served as a reliable indicator of the presence of mycolactone-producing mycobacteria in a water body, and there was no significant difference between fish and amphibian positivity rates (P-value=0.106). There was a significant difference between water bodies in the total number of ER-positive specimens (P-value=0.0164). CONCLUSIONS: Although IS2404-positive tadpoles and fish have been reported, this is the first VNTR confirmation of M. ulcerans or M. liflandii in wild amphibian and fish populations in West Africa. Results from this study suggest that amphibians should be carefully examined as potential reservoirs for M. ulcerans in West Africa, and that H. bimaculatus may be useful as an indicator of habitats likely to support mycolactone-producing mycobacteria.

Spatial and temporal variability of macroinvertebrates in spawning and non-spawning habitats during a salmon run in Southeast Alaska.

Spawning salmon create patches of disturbance through redd digging which can reduce macroinvertebrate abundance and biomass in spawning habitat. We asked whether displaced invertebrates use non-spawning habitats as refugia in streams. Our study explored how the spatial and temporal distribution of macroinvertebrates changed during a pink salmon (Oncorhynchus gorbuscha) spawning run and compared macroinvertebrates in spawning (riffle) and non-spawning (refugia) habitats in an Alaskan stream. Potential refugia included: pools, stream margins and the hyporheic zone, and we also sampled invertebrate drift. We predicted that macroinvertebrates would decline in riffles and increase in drift and refugia habitats during salmon spawning. We observed a reduction in the density, biomass and taxonomic richness of macroinvertebrates in riffles during spawning. There was no change in pool and margin invertebrate communities, except insect biomass declined in pools during the spawning period. Macroinvertebrate density was greater in the hyporheic zone and macroinvertebrate density and richness increased in the drift during spawning. We observed significant invertebrate declines within spawning habitat; however in non-spawning habitat, there were less pronounced changes in invertebrate density and richness. The results observed may be due to spawning-related disturbances, insect phenology, or other variables. We propose that certain in-stream habitats could be important for the persistence of macroinvertebrates during salmon spawning in a Southeast Alaskan stream.

Detection of Mycobacterium ulcerans in the environment predicts prevalence of Buruli ulcer in Benin.

BACKGROUND: Mycobacterium ulcerans is the causative agent of Buruli ulcer (BU). In West Africa there is an association between BU and residence in low-lying rural villages where aquatic sources are plentiful. Infection occurs through unknown environmental exposure; human-to-human infection is rare. Molecular evidence for M. ulcerans in environmental samples is well documented, but the association of M. ulcerans in the environment with Buruli ulcer has not been studied in West Africa in an area with accurate case data. METHODOLOGY/PRINCIPAL FINDING: Environmental samples were collected from twenty-five villages in three communes of Benin. Sites sampled included 12 BU endemic villages within the Ouheme and Couffo River drainages and 13 villages near the Mono River and along the coast or ridge where BU has never been identified. Triplicate water filtrand samples from major water sources and samples from three dominant aquatic plant species were collected. Detection of M. ulcerans was based on quantitative polymerase chain reaction. Results show a significant association between M. ulcerans in environmental samples and Buruli ulcer cases in a village (p = 0.0001). A "dose response" was observed in that increasing numbers of M. ulceran- positive environmental samples were associated with increasing prevalence of BU cases (R(2) = 0.586). CONCLUSIONS/SIGNIFICANCE: This study provides the first spatial data on the overlap of M. ulcerans in the environment and BU cases in Benin where case data are based on active surveillance. The study also provides the first evidence on M. ulcerans in well-defined non-endemic sites. Most environmental pathogens are more broadly distributed in the environment than in human populations. The congruence of M. ulcerans in the environment and human infection raises the possibility that humans play a role in the ecology of M. ulcerans. Methods developed could be useful for identifying new areas where humans may be at high risk for BU.

Effectiveness of wound cleansing treatments on maggot (Diptera, Calliphoridae) mortality.

Myiasis is defined as an infestation of the organs and/or tissues of human and other animals by fly maggots. Fly species that normally breed in meat or carrion (Diptera: Calliphoridae, Sarcophagidae) may become involved in cutaneous myiasis by colonizing preexisting wounds. Reports of human wound myiasis contracted in hospitals and nursing homes, especially when patients are chronically ill or bed-ridden, are not uncommon across North America and often result in cases of neglect and civil litigation. Based on a case history dealing with this latter situation and circumstances surrounding the treatment of maggot infestation, we designed an experiment to assess the effectiveness of wound cleansing solutions on maggot mortality. Treatments, consisting of four commonly used cleaning solutions (isopropyl alcohol, Dakin's solution, iodine, and hydrogen peroxide) and a control (deionized water), were applied to experimental units (n=5), with each unit consisting of groups of actively feeding Lucilia sericata maggots (Diptera: Calliphoridae). Every 24h, treatments were applied and mortality was assessed for the duration of the study (14 days). Total mean mortality increased over the duration of the experiment, with an initial large increase (10-25%) after the first treatment application, followed by a gradual increase over the remainder of the study. General differences among treatments indicated greatest mean total mortality for Dakin's solution (sodium hypochlorite) (46%), followed by isopropyl alcohol (42%), Betadine (37%), hydrogen peroxide (33%) and lowest mortality for the control (25%); however, no statistically significant differences were observed among treatments and no treatment resulted in 100% maggot mortality. Traditional wound cleansing solutions may not be sufficient for maggot infestations of pre-existing wounds and supplemental treatments may be necessary to effectively treat cases of wound myiasis.