A survey of Wolbachia infection in brachyceran flies from Iran.

Wolbachia is a maternally inherited intracellular bacterium that is considered to be the most plentiful endosymbiont found in arthropods. It reproductively manipulates its host to increase the chances of being transmitted to the insect progeny; and it is currently used as a means of suppressing disease vector populations or controlling vector-borne diseases. Studies of the dissemination and prevalence of Wolbachia among its arthropod hosts are important for its possible use as a biological control agent. The molecular identification of Wolbachia relies on different primers sets due to Wolbachia strain variation. Here, we screened for the presence of Wolbachia in a broad range of Brachycera fly species (Diptera), collected from different regions of Iran, using nine genetic markers (wsp, ftsZ, fbpA, gatB, CoxA, gltA, GroEL dnaA, and 16s rRNA), for detecting, assessing the sensitivity of primers for detection, and phylogeny of this bacterium. The overall incidence of Wolbachia among 22 species from six families was 27.3%. The most commonly positive fly species were Pollenia sp. and Hydrotaea armipes. However, the bacterium was not found in the most medically important flies or in potential human disease vectors, including Musca domestica, Sarcophaga spp., Calliphora vicinia, Lucilia sericata, and Chrysomya albiceps. The primer sets of 16s rRNA with 53.0% and gatB with 52.0% were the most sensitive primers for detecting Wolbachia. Blast search, phylogenetic, and MLST analysis of the different locus sequences of Wolbachia show that all the six distantly related fly species likely belonging to supergroup A. Our study showed some primer sets generated false negatives in many of the samples, emphasizing the importance of using different loci in detecting Wolbachia. The study provides the groundwork for future studies of a Wolbachia-based program for control of flies.

Colonization pattern and thermal needs of immature phases of Sarcophaga argyrostoma (Diptera: Sarcophagidae): Significance for estimating postmortem interval.

Members of Sarcophagidae are necrophagous and are commonly found on decaying carcasses; and their developmental forms are important indicators for the approximation of lowest postmortem interval (PMImin). This work describes the biological characteristics of Sarcophaga argyrostoma from Tehran, Iran. Various temperature regimes were applied to estimate the thermal summation constant (k) and thermal requirements for development of S. argyrostoma. Five growth proceedings, containing 1st ecdysis, 2nd ecdysis, wandering, pupariation and eclosion, were investigated under eight fixed temperature regimes (6-30 °C). The effects of fly age, freshness, and availability of oviposition substrate on oviparity and viviparity was studied. At 6 °C, no development occurs, and at 8 °C only the first ecdysis occurs. At temperatures between 10 and 30 °C, all immature stages proceeded to the adult stage, and thus immature development was analyzed at these six remaining temperatures. The development phases needed minimum (Dz ± SE) 5.4(0.4), 8.5(0.26), 5.3(0.44), 3.8(0.1), and 6.6 (0.6)°C to attain one of the succeeding developmental occasion, correspondingly. The approximated K for those five occasions were 15.04 ± 1.12, 12.62 ± 0.65, 140.36 ± 4.35, 14.59 ± 0.6, and 222.8 ± 4.18°-day (DD) accordingly. When the food substrate is available and fresh, the female prefers to lay eggs, no matter how old she is. However, the chance of ovoviviparity increased when no oviposition substrate was available. The truth that S. argyrostoma able to either larviposit or to lay eggs encompasses serious consequences for the precise estimation of the PMImin, as the existence of larvae resulting from eggs laid on the carcass could add hours (based on ambient temperature) to the PMImin.

Scalp myiasis associated with soft tissue sarcoma lesion: a case report and review of relevant literature.

BACKGROUND: Sarcophagidae is one of the main fly families that is attracted to open wounds, ulcers, lesions, and other injuries for depositing their larvae. The presence of larvae of flies in human tissues makes myiasis. Myiasis on the scalp could be more frightening in comparison with myiasis on the other parts of the body. It is a rare myiasis case that shows the ability of myiasis agents to attack various parts of the body. On the other hand, reporting of myiasis cases by Sarcophagidae larvae is not common due to difficulties in their identification. This study aimed to emphasize the importance of Sarcohagidae larvae in producing myiasis by describing the first case of soft tissue sarcoma infestation and provides a review of human myiasis by larvae of the Sarcophagidae family during 2010-2023 and also a review of wound myiasis cases associated with malignancy during 2000-2023. CASE PRESENTATION: A case of sarcoma cancer myiasis is reported on the scalp of a 43-year-old man who referred to one of Tehran's hospitals for surgical treatment of cancer. Before surgery, insect larvae were observed in the area of ​​sarcoma. The larvae were isolated, examined morphologically, and identified as Sarcophaga spp. CONCLUSIONS: Myiasis has been considered as a neglected disease. Publishing of myiasis cases could be useful to alert health policy-makers about its danger and appearance in the community. It is not usual but can be expected even on the scalp of the human head. Exact daily supervision and dressing of the wound could be recommended to prevent cutaneous myiasis.

Larval therapy vs conventional silver dressings for full-thickness burns: a randomized controlled trial.

BACKGROUND: This is the first clinical trial to investigate the effectiveness of maggot debridement therapy (MDT) for full-thickness burn injuries in comparison to conventional silver dressings. METHODS: Thirty-one cases with full-thickness (grade III based on ICD-10 classifications version 2019) burns were assigned into larval therapy (15 cases) and conventional treatment (16 cases) groups. Participants in the MDT group have received loose larvae on days 0, 2, 4, and 6, while controls received a conventional regimen comprised of sharp debridement, silver sulfadiazine, antibiotic therapy, and offloading every day. The primary and secondary outcomes were defined as the time to debridement (from admission to skin autograft) and time to healing (from admission to complete healing post-skin autograft). Patients in two groups were also compared in terms of necrosis resolution, granulation, and granulation/necrosis (g/n) ratio during study time periods. RESULTS: Participants who received larvae had significantly decreased necrosis on days 2 (p = 0.028) and 4 (p = 0.023) compared to those who received control treatment. Significant differences (p < 0.001) were also observed for granulation between the two groups in favor of MDT and the fold changes of g/n in the larvae group were 5, 15, and 13 times higher than that for the conventional regimen on days 2, 4, and 6 of treatment, respectively. Strikingly, a subgroup analysis of high necrotic burns (necrosis > 50%) revealed a significant improvement (p < 0.001) for MDT compared to the control treatment. There were also significant differences (p < 0.001) for the time to debridement and time to healing between the two groups. However, bacterial contamination did not show significant changes between the two treatment regimens. CONCLUSIONS: Our findings revealed that MDT has a favorable superiority over conventional regimen for the treatment of grade-III burns, and thus further clinical trials with larger sample size are warranted to confirm these results.

Evaluating leishmanicidal effects of Lucilia sericata products in combination with Apis mellifera honey using an in vitro model.

Leishmaniasis is a zoonotic disease caused by an intracellular parasite from the genus Leishmania. Lack of safe and effective drugs has increasingly promoted researches into new drugs of natural origin to cure the disease. The study, therefore, aimed to investigate the anti-leishmanial effects of Lucilia sericata larval excretion/secretion (ES) in combination with Apis mellifera honey as a synergist on Leishmania major using an in vitro model. Various concentrations of honey and larval ES fractions were tested against promastigotes and intracellular amastigotes of L. major using macrophage J774A.1 cell line. The inhibitory effects and cytotoxicity of ES plus honey were evaluated using direct counting method and MTT assay. To assess the effects of larval ES plus honey on the amastigote form, the rate of macrophage infection and the number of amastigotes per infected macrophage cell were estimated. The 50% inhibitory concentration (IC50) values were 21.66 µg/ml, 43.25 60 µg/ml, 52.58 µg/ml, and 70.38 µg/ml for crude ES plus honey, ES >10 kDa plus honey, ES <10 kDa plus honey, and honey alone, respectively. The IC50 for positive control (glucantime) was 27.03 µg/ml. There was a significant difference between viability percentages of promastigotes exposed to different doses of applied treatments compared to the negative control (p≤ 0.0001). Microscopic examination of amastigote forms revealed that dosages applied at 150 to 300 µg/ml significantly reduced the rate of macrophage infection and the number of amastigotes per infected macrophage cell. Different doses of larval products plus honey did not show a significant toxic effect agaist macrophage J774 cells. The larval ES fractions of L. sericata in combination with A. mellifera honey acted synergistically against L. major.

Sex-typing of ingested human blood meal in Anopheles stephensi mosquito based on the amelogenin gene.

Identifying the sex of human hosts of insect disease vectors, using PCR amplification of the amelogenin gene (AMEL) from the ingested blood meal is an increasingly useful technique for epidemiological studies of vector-borne diseases, as well as within the criminal justice system. Detection of DNA from ingested blood is influenced by the choice of DNA extraction method, genomic target region, type and length of PCR, and rate of degradation in the DNA samples over time. Here, we have tested two types of PCR (i.e. conventional and nested), producing differently-sized PCR products, in time-course assays targeting the human AMEL gene in Anopheles stephensi mosquitoes that were fed on human male and female blood. The fed female mosquitoes were allowed to digest at 28 °C for times ranging from 0 to 120 h. Three AMEL primer pairs were used to amplify three sequences that were 977, 539, and 106 bp for the X chromosome and 790, 355, and 112 bp for Y. We found that time since feeding had a significant negative effect on the success of PCR amplification. The shortest fragments (106 and 112 bp) were amplified for the longest time after blood feeding (up to 60 h), whereas the medium and longest loci were not amplified by conventional PCR even at 0 h. However, the nested PCR protocol, targeting the medium sequence, could detect small amounts of human DNA up to 36 h (1.5 days) after the blood meal. The shortest PCR assay standardized herein successfully detected small amounts of human DNA in female mosquitoes up to 60 h after the blood meal. This assay represents a promising tool for identifying the sex of the human host from the blood meal in field-collected female mosquitoes.

The therapeutic effect of larval saliva and hemolymph of Lucilia sericata on the treatment of Leishmania major lesion in BALB/c mice946.

BACKGROUND: Treatment of cutaneous leishmaniasis (CL) remains a major challenge for the public health and medical community. It has been claimed that natural compounds derived from fly larvae have anti-leishmania properties against some species of Leishmania. The present study aimed at assessing the in vitro effects of larval products of Lucilia sericata against the promastigote and intracellular amastigote forms of Leishmania major. Also, the therapeutic effect of larval products on lesions induced by L. major infection was evaluated in BALB/c mice models. METHODS: Parasite specimens and macrophage cells were exposed to varying concentrations of larval products for 24-120 h. Lesion progression and parasite load were investigated in the models to assess the therapeutic effects of the products. RESULTS: The larval products displayed more potent cytotoxicity against L. major promastigotes. The IC50 values for larval saliva and hemolymph were 100.6 and 37.96 ug/ml, respectively. The IC50 of glucantime was 9.480 ug/ml. Also, the saliva and hemolymph of L. sericata exhibited higher cytotoxicity against the promastigotes of L. major but were less toxic to the macrophage cells. Treatment with leishmanicidal agents derived from larvae of L. sericata decreased the infection rate and the number of amastigotes per infected host cell at all concentrations. Lesion size was significantly (F (7, 38) = 8.54, P < 0.0001) smaller in the treated mice compared with the untreated control group. The average parasite burden in the treated mice groups (1.81 ± 0.74, 1.03 ± 0.45 and 3.37 ± 0.41) was similar to the group treated with a daily injection of glucantime (1.77 ± 0.99) and significantly lower (F (7, 16) = 66.39, P < 0.0001) than in the untreated control group (6.72 ± 2.37). CONCLUSIONS: The results suggest that the larval products of L. sericata were effective against L. major parasites both in vivo and in vitro. However, more clinical trial studies are recommended to evaluate the effects of these larval products on human subjects.

Maggot debridement therapy and complementary wound care: a case series from Nigeria.

OBJECTIVE: Maggot debridement therapy (MDT) is an emerging procedure involving the application of sterile maggots of the Dipteran species (commonly Lucilia sericata) to effect debridement, disinfection and promote healing in wounds not responding to antimicrobial therapy. Data on MDT in sub-Saharan Africa (including Nigeria) are scarce. This study aimed to use medicinal grade maggots as a complementary method to debride hard-to-heal necrotic ulcers and thereby promote wound healing. METHOD: In this descriptive study, we reported on the first group of patients who had MDT at Aminu Kano Teaching Hospital (AKTH), a tertiary hospital in northern Nigeria. The first instar larvae of Lucilia sericata were applied using the confinement (free-range) maggot therapy dressing method under aseptic conditions. RESULTS: Diabetic foot ulcer (DFU) grade III-IV constituted more than half of the wounds (53.3%), followed by necrotising fasciitis (30%), and post-traumatic wound infection (10%). Others (6.7%, included pyomyositis, surgical site infection and post traumatic wound infection). The median surface area of the wounds was 56cm2. Of the 30 patients, half (50%) had two MDT cycles with a median time of four days. Of the wounds, 22 (73%) were completely debrided using maggots alone while eight (27%) achieved complete debridement together with surgical debridement. Wound culture pre-MDT yielded bacterial growth for all the patients and Staphylococcus aureus was the predominant isolate in 17 wounds (56.7%) while Pseudomonas aeruginosa and Streptococcus pyogenes were predominant in five wounds (16.7%) each. Only four (13.3%) wound cultures yielded bacterial growth after MDT, all Staphylococcus aureus. CONCLUSION: A good prognosis was achieved post-MDT for various wounds. MDT effectively debrides and significantly disinfects wounds involving different anatomical sites, thus enhancing wound healing and recovery. MDT is recommended in such wounds.

Wing measurements are a possible tool for the identification of European forensically important Sarcophagidae.

The flesh flies are a group of insects well known for their forensic importance. Reliable identification of these flies relies on the use of either molecular markers or the morphology of the male genital apparatus. Identification of female flesh flies is more time consuming and less reliable than their male counterparts. This is particularly problematic for forensic entomology practitioners, because female flesh flies are more abundant than males in carrion arthropod assemblages. As such, it is critical that alternative methods for flesh fly identification are established that are equally effective for both sexes. One promising technique is the use of wing measurements, which have been shown to be reliable for the identification of some groups of necrophagous Diptera from several geographical regions. We applied this method to the European Sarcophagidae for the first time, using a dataset of 881 specimens representing 29 species and 5 genera. Species identifications were based on 15 landmarks located at wing vein junctions. We also combined our results with data from previous studies of Calliphoridae and Muscidae which utilised the same methodology, enabling the testing of family level identification using wing morphometrics. Species identifications using wing measurements had varied success. While some species were successfully identified without error, others, particularly from the genus Sarcophaga, were often misclassified. Notably, in several species wing measurements successfully identified both males and females. The results presented here suggest that wing measurements are a promising complementary method to other methods for the identification of necrophagous Sarcophagidae especially in material unsorted at the family level. It can also be used to double check identification performed by a taxonomist using traditional methods.

A Comparative Study on the Biodiversity and Species Richness of Phlebotomine Sand Flies (Diptera: Psychodidae) in Kermanshah and Khuzestan Provinces of Iran.

Background: Leishmaniasis is one of the most important vector-borne and neglected tropical diseases in many parts of the world. The main objective of this study was to determine the biodiversity of phlebotomine sand flies (Diptera: Psychodidae, Phlebotominae) in Khuzestan and Kermanshah Provinces of Iran. Methods: Sampling was conducted in Khuzestan and Kermanshah Provinces using sticky paper traps and CDC light traps. The samples were then stored in 96% alcohol-containing vials, mounted, and identified. Afterward, the alpha diversity (using Simpson's, Shannon-Weiner's diversity, evenness, Maghalef's, Menhinick's, Hill N 1 and N 2 indices) and beta diversity indices (using Sorensen's and Jaccard's coefficients) were calculated. Results: A total of 4302 sand flies were caught and identified which comprised mostly of Phlebotomus papatasi, Ph. alexandri and Ph. sergenti. It was found that among the four counties of Ahvaz, Shush, Shushtar and Dezful in Khuzestan Province, Shush had the lowest, and Shushtar, the highest amount of species diversity and evenness. In the four studied counties of Kermanshah Province, species diversity in Kermanshah County had the lowest amount and Sarpol-e-Zahab, the highest. The species richness in Kermanshah County was the lowest, while Qasr-e-Shirin County had the highest amount. Conclusion: An overview of the biodiversity of phlebotomine sand flies in Kermanshah County in Kermanshah Province, and Shush County in Khuzestan Province, showed less stability in community structure of these vectors, which can be counted as an alarm for emerging dominant vectors among the studied counties and have the potential to increase the prevalence of leishmaniasis.

A Parallel Randomized Clinical Trial for Comparison of Two Methods of Maggot Therapy, Free-Range Larvae and Larval-bag, in Diabetic Ulcer (Wagner 2).

In the present study, the efficiency of two formulations of maggot therapy: free-range larvae on the wounds and larvae in a special bag (larval-bag), was compared for healing diabetic ulcers with Wagner 2 diabetic ulcer. This study was conducted as a parallel randomized clinical trial. Out of 281 patients with Wagner grade 2 diabetic ulcers referred to the wound clinic, 54 patients who met the inclusion criteria were randomly assigned to intervention groups. The disinfected larvae of Lucilia sericata were put on the wounds with 2 methods, free-range larvae and larval-bag. Follow up was done at every 48 h interval until the full appearance of granulated tissues. The main measures were wound bed preparation, removing of necrotic tissues, appearing of granulated tissues, and removing of bacterial infections in the wounds. Statistical analysis based on the Kaplan-Meier curve and the Wilcoxon (Breslow) test showed a significant reduction in wound healing time by using free-range larvae in comparison with larval-bag (P = .03). The median time to debridement was 4 days in the free-range larval group (95% confidence interval: 3-9 days) while it was 9 days in the larval-bag group (95% confidence interval: 5-16 days). Debridement rate (proportion of removed necrotic tissue surfaces) at any time in the free-ranged larvae group was 1.78 times that of the bagged larvae group (95% confidence interval 1.01-3.15, P = .036). There was no significant difference between free-range larval use and larval-bag in the acceptability of maggot therapy by patients (P = .48). It can be concluded that both of two formulations of the larval therapy (free-range larvae and larval-bag) could be recommended for cleaning out and bed preparation of diabetic ulcers. However, using free-range larvae is hardly recommended on the wounds which are eligible to use.

Thermal requirements of immature stages of Chrysomya albiceps (Diptera: Calliphoridae) as a common forensically important fly.

Entomological material may be used to estimate the time since death occurred (postmortem interval, PMI) in forensically obscure cases. The method that is commonly used to calculate minimum post-mortem interval (mPMI, i.e., the least amount of time since one can be confident death occurred) is based on the relationship between insect development and ambient temerature. Isomegalen and isomorphen diagrams are among methods allowing to calculate the age of necorphagous insects, yet thermal summation models provide the most precise and acurate estimations. The digrams are prepared based on the length or the developmental stages of the larvae as a function of time and mean ambient temperature. A knowledge of thermal requirements, in particular lower temperature threshold (Dz) at which development of a species terminates, is of essential importance to calculate ADD (Accumulated Degree Days). In this study different temperature regimes were used to construct the isomorphen diagram, examinate changes in larval body length at different ambient temperatures and to estimate the thermal requirements for developemnt of Chrysomya albiceps, the most common dipteran species reported on human and animal cadavers in Iran. Six development events including hatching, 1st ecdysis, 2nd ecdysis, wandering, pupariation and eclosion were studied under eleven constant temperature regims (17-37 0C). The development rate of Ch. albiceps increased as temperature increased. The larval length peaked at the end of third stage and then decreased at wandering stage. The maximum larval length occurred at 72 h post oviposition at either 31, 33, or 35 °C. At 17 °C, larvae did not hatch from eggs and at 37 °C wandering larvae did not proceed to pupariation, and thus larval development were analysed at the nine left over temperatures. The development stages required at least (Dz ± SE) 13.04 ± 0.37, 14.29 ± 0.45, 15.69 ± 0.56, 15.18 ± 0.56, 14.94 ± 0.48, and 11.23 ± 0.41 °C to reach one of the successive developmentl events, respectively. The estimated thermal summation constant (k) for those the six events were 10.43 ± 0.27, 19.31 ± 0.32, 27.87 ± 1.3, 55.94 ± 1.82, 66.69 ± 3.5, and 143.52 ± 5.61 ADD accordingly.

The leishmanicidal effect of Lucilia sericata larval saliva and hemolymph on in vitro Leishmania tropica.

BACKGROUND: Leishmaniasis is a major parasitic disease worldwide, except in Australia and Antarctica, and it poses a significant public health problem. Due to the absence of safe and effective vaccines and drugs, researchers have begun an extensive search for new drugs. The aim of the current study was to investigate the in vitro leishmanicidal activity of larval saliva and hemolymph of Lucilia sericata on Leishmania tropica. METHODS: The effects of different concentrations of larval products on promastigotes and intracellular amastigotes of L. tropica were investigated using the mouse cell line J774A.1 and peritoneal macrophages as host cells. The 3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and direct observation and counting method were used to assess the inhibitory effects and cell cytotoxicity of the larval products. The effects of larval products on the amastigote form of L. tropica were quantitatively estimated by calculating the rate of macrophage infection, number of amastigotes per infected macrophage cell, parasite load and survival index. RESULTS: The 50% cytotoxicity concentration (CC50) value of both larval saliva and hemolymph was 750 µg/ml, and the 50% inhibitory concentration (IC50) values were 134 µg/ml and 60 µg/ml for larval saliva and larval hemolymph, respectively. The IC50 for Glucantime, used a positive control, was (11.65 µg/ml). Statistically significant differences in viability percentages of promastigotes were observed for different doses of both larval saliva and hemolymph when compared with the negative control (p ≤ 0.0001). Microscopic evaluation of the amastigote forms revealed that treatment with 150 µg/ml larval hemolymph and 450 µg/ml larval saliva significantly decreased the rate of macrophage infection and the number of amastigotes per infected macrophage cell. CONCLUSION: Larval saliva and hemolymph of L. sericata have acceptable leishmanicidal properties against L. tropica.

Evaluation of Susceptibility of Aedes caspius (Diptera: Culicidae) to Insecticides in a Potent Arboviral-Prone Area, Southern Iran.

BACKGROUND: Southern part of the country is a high risk for mosquito transmitted Arboviruses. This study was carried out to determine the base line susceptibility of the Aedini mosquitoes to the WHO-recommended insecticide. METHODS: Larval collection was carried out by dipping method and adult collection occurred by suction tube from January to December 2017. The adult susceptibility test was assessed to Bendiocarb 0.1%, DDT 4%, Deltamethrin 0.05%, Lambda-cyhalothrin 0.05%, Malathion 5% and, Permethrin 0.75% at different interval times as well as at discriminative dose recommended by WHO. The larval susceptibility test was occurred using Temephos and Bacillus thuringiensis serotype H-14, at different concentrations. The LT50, LT90 and LC50, LC90 values were calculated for plotting the regression line using Microsoft office Excel software ver. 2007. RESULTS: Aedes caspius was quite resistant to DDT, Malathion, Bendiocarb and showed susceptible or tolerant to other insecticides.The LT50 and LT90 values to DDT in this species were 157.896, and 301.006 minutes, respectively. The LC50 and LC90 values of Ae. caspius to Temephos were 0.000068, and 0.000130ppm, the figures for B. thuringiensis was 111.62 and 210.2ppm, respectively. CONCLUSION: A routine and continuous study for monitoring and evaluation of different species of Aedes to insectides is recommend at different parts of country for decision making.

Using a combination therapy to combat scalp necrosis: a case report.

BACKGROUND: Chronic nonhealing wounds are very expensive to treat and debilitating, and they reduce health-related quality of life. Scalp necrosis is very rare due to its rich vascularity. However, any post-traumatic wounds with secondary infection can lead to scalp necrosis. CASE PRESENTATION: We report a case of a 77-year-old Azerbaijani man with a history of diabetes who had a car accident and sustained a scalp wound. He underwent reconstructive surgery for the scalp wound. The wound became infected, and scalp necrosis developed following the surgery. There was no progress in wound healing in spite of conventional wound therapy. We combined maggot debridement therapy with negative-pressure wound therapy and amniotic membrane grafting for 7 months. Necrotic tissues began to be eliminated after the second use of larva therapy, and the wound became free of necrotic tissues with clear increase of granulated tissues after four treatments with maggot debridement therapy. Then, we applied negative-pressure wound therapy and amniotic membrane grafting to accelerate wound healing and improve wound closure. The patient's scalp wound recovered well, and he was discharged to home in good condition. CONCLUSIONS: Medical and wound care teams can benefit from this combination therapy when dealing with nonhealing necrotic wounds.

Relationship Between the Distribution and Biodiversity of Sand Flies (Diptera: Psychodidae) With the Incidence of Zoonotic Cutaneous Leishmaniasis in Endemic Foci of Golestan Province, Iran.

Zoonotic cutaneous leishmaniasis (ZCL) is prevalent in Golestan Province, Iran. The current study determined the relationship between the distribution and biodiversity of sand flies with cutaneous leishmaniasis at 14 villages in plain and hillsides areas. In each village from July to September 2017, 60 sticky traps and 2 CDC light traps were laid. Spearman and Mann-Whitney tests were used to determine the relationship between the incidence of ZCL and the abundance of different species of sand flies. Simpson, Shannon-Wiener, Evenness, and Margalef indices were calculated to estimate the diversity of species. A total of 5,295 phlebotomine sand flies were collected, comprising 10 species of the genus Phlebotomus (3,947 flies) and 7 species of genus Sergentomyia (1,248 flies). The abundance of sand flies and incidence of ZCL in plain areas were greater than that of hillsides areas (P = 0.013, P = 0.002). There was a significant correlation between the incidence of ZCL and the abundance of Phlebotomus papatasi (r = 0.72, P = 0.004) and P. caucasicus groups (P = 0.006; 0.022). In the Shannon-Wiener index, the rest of the biodiversity indices were reduced in higher-altitude areas. Increasing Shannon-Wiener index showed higher diversity of sand flies in higher-altitude areas. Data of the reported cases of leishmaniasis in plain areas can reveal the relationship between less diversity index (Shannon-Wiener), higher dominant diversity index (Simpson), and incidence of leishmaniasis in these areas.

Molecular Species Identification of Six Forensically Important Iranian Flesh Flies (Diptera).

BACKGROUND: Flesh flies (Diptera: Sarcophagidae) are considered as myiasis agents and important evidences in forensic investigations. However, their use has been restricted because, at all larval stages and female adults, morphological species identification is difficult or very challenging. This study investigated to test utility of mitochondrial cytochrome oxidase subunit I (mt-COI) sequences for differentiation of six forensically important Iranian flesh flies namely, Sarcophaga crassipalpis, S. flagellifera, S. hirtipes, S. aegyptica, S. africa and S. argyrostoma. METHODS: Male specimens were morphologically identified to species level and then the genomic DNA of the flies were extracted and subjected to polymerase chain reaction (PCR) against mt-COI gene. The PCR products were sequenced and the obtained sequences were analyzed for the species specific restriction fragment length polymorphisms (RFLPs). RESULTS: Rate of genetic variation between species was 6-10% which was enough to find restriction enzymes (RE) that were able to produce species-specific RFLP profiles. Combinations of three REs: BsrFI, RsaI and HinfI, provided diagnostic bands for identification of the six Sarcophaga species. CONCLUSION: The results of this study showed that molecular markers such as RFLPs enhancing the use of evidence from flesh flies in forensic investigation. However, lack proper restriction sites in the COI region inhibited introduction of a single restriction enzyme for easy species identification. It is recommended to apply larger part of DNA such as combination of COI and COII genes to provide better RFLP markers for species identification of flesh flies.

Biodiversity of Medically Important Calyptratae Flies (Diptera: Schizophora) in Hospitals in the Northern Coastline of the Persian Gulf, Iran.

Domestic filth flies play a significant role in the transmission of nosocomial infections and cause nosocomial myiasis in hospitals worldwide. This descriptive cross-sectional study was conducted in five hospitals of the Hormozgan province, of southern Iran. Adult flies were collected by bottle fly traps. Specimens were pinned and morphologically identified using the relevant keys. Species biodiversity indices including Menhinick (M), Shannon-Wiener (H'), Pielou's Evenness (E), and Simpson's diversity (D) were calculated. Totally 2,298 individuals of 18 species of flies belonging to nine genera and four families were collected. Chrysomya megacephala was the most frequent species (37.3%), followed by Chrysoma albiceps (24.8%), Fannia canicularis (5.1%), Musca domestica (4.8%), Sarcophaga aegyptica (3.7%), and 15 other species (24.4%). Our results show a high diversity and abundance of domestic filth flies in the studied hospitals. Relatively similar data derived from various hospitals in this study can show the uniformity in sanitary conditions of hospitals. On the other hand, estimation of diversity indices alarmed for potential of dominant species in fly communities in hospital environments.

Muscle attachment site patterns for species determination in West Palaearctic Wohlfahrtia (Diptera: Sarcophagidae) of medical and veterinary importance.

The flesh fly genus Wohlfahrtia Brauer & Bergenstamm contains at least six species of medical and veterinary importance. Traditional methods of species identification in specimens of Wohlfahrtia, however, are restricted mostly to adult forms. Muscle attachment site (MAS) patterns allow for species determination in larval forms. MAS patterns in third instar larvae of six common West Palearctic species of Wohlfahrtia have been analyzed for this study. As in previously investigated Calliphoridae and Sarcophagidae, MAS patterns were found to be species specific. A genus pattern was established to be used as base for comparison in further species determination. For the first time a tool is provided for species identification of such broad range in larvae of Wohlfahrtia species. Wohlfahrtia patterns are composed of a significantly higher number of MAS than patterns found in Sarcophaga. Specifics of the six species analyzed are explained in detail. The larvae of the well-known species W. magnifica, an obligate traumatic myiasis agent, had to be excluded from the analysis as a great number of spines on the outside obscure muscle attachment sites on the inside of the cuticle.