Machine learning analysis of wing venation patterns accurately identifies Sarcophagidae, Calliphoridae and Muscidae fly species.

In medical, veterinary and forensic entomology, the ease and affordability of image data acquisition have resulted in whole-image analysis becoming an invaluable approach for species identification. Krawtchouk moment invariants are a classical mathematical transformation that can extract local features from an image, thus allowing subtle species-specific biological variations to be accentuated for subsequent analyses. We extracted Krawtchouk moment invariant features from binarised wing images of 759 male fly specimens from the Calliphoridae, Sarcophagidae and Muscidae families (13 species and a species variant). Subsequently, we trained the Generalized, Unbiased, Interaction Detection and Estimation random forests classifier using linear discriminants derived from these features and inferred the species identity of specimens from the test samples. Fivefold cross-validation results show a 98.56 ± 0.38% (standard error) mean identification accuracy at the family level and a 91.04 ± 1.33% mean identification accuracy at the species level. The mean F1-score of 0.89 ± 0.02 reflects good balance of precision and recall properties of the model. The present study consolidates findings from previous small pilot studies of the usefulness of wing venation patterns for inferring species identities. Thus, the stage is set for the development of a mature data analytic ecosystem for routine computer image-based identification of fly species that are of medical, veterinary and forensic importance.

Molecular identification of Eimeria hestermani and Eimeria prionotemni from a red-necked wallaby (Macropodidae; Macropus rufogriseus) in Japan.

To date, more than 50 Eimeria spp. have been isolated from marsupials of the family Macropodidae. Although 18 species of Eimeria have been previously detected from multiple animal species belonging to the genus Macropus of the family, limited genetic analyses of the parasites are available, and their pathogenicity remains unclear. Here, we report the isolation of Eimeria spp. from a zoo specimen of red-necked wallaby (Macropodidae; Macropus rufogriseus). Specifically, two distinct types of Eimeria oocysts were recovered, one from the feces before treatment with an anthelmintic and the second from the intestinal contents after death of the animal. The oocysts obtained from the two sources were morphologically identified as E. hestermani and E. prionotemni, respectively. We successfully determined partial gene sequences from the two isolates, including segments of the 18S rRNA genes, and for the first time have used phylogenetic analyses of these sequences to assign the species to distinct clades. In combination with further genetic data, these results are expected to help elucidate the pathogenicity and host ranges of Eimeria spp. within the respective family and genus.

Occurrence and genetic identifications of porcine Entamoeba, E. suis and E. polecki, at Tangerang in West Java, Indonesia.

Entamoeba suis and E. polecki subtype (ST) 1 and ST3 recently have been inferred to be virulent in pigs. However, because relevant molecular epidemiological surveys have been limited, the prevalences of these species remain unknown and their pathogenicities are still controversial. We surveyed 196 fecal samples of pigs (118 of adults, 78 of piglets) at Tangerang in West Java, Indonesia, in 2017, employing PCR using porcine Entamoeba-specific primers. E. suis was the more frequently detected species, observed in 81.1% of samples, while E. polecki ST1 and ST3 were detected in 18.4% and 17.3% of samples, respectively; mixed infections (harboring 2-3 species or subtypes of Entamoeba) were confirmed in 29.3% of positive samples. Statistically significant differences in the positive rates were not seen between adult pigs and piglets, except for those of E. polecki ST3. The prevalences of Eimeria spp. and/or Cystoisospora suis (79.1%), strongyles (55.6%), and Strongyloides spp. (6.1%) were also observed morphologically in the samples. Further chronological or seasonal investigations of pigs and humans in these high-prevalence areas are needed to assess the virulence of the Entamoeba parasites, including the effects on pig productivity, and to evaluate the zoonotic impacts of these organisms.

Ectoparasites Infestation to Small Ruminants and Practical Attitudes among Farmers toward Acaricides Treatment in Central Region of Java, Indonesia.

Ectoparasite infestations are one of the major problems affecting goat and sheep farming. Disease resulting from these infestations can cause changes in physical appearance, such as severe lesions on the skin, and economic consequences in the form of significantly reduced selling prices. This study aimed to determine the prevalence of ectoparasites in the Boyolali district, Central Java, Indonesia. A total of 651 sheep and goats were surveyed in this study. The parasites were collected via skin scraping, twister, or manually from clinically infected goats and sheep in traditional farms. All of the ectoparasites collected were successfully identified. The prevalence of ectoparasites in ruminants in Boyolali was 97.8% (637/651). The species make-up was as follows: Bovicola caprae 97.8% (637/651), Linognathus africanus 39% (254/651), Haemaphysalis bispinosa 3.5% (23/651), Ctenocephalides spp. 0.2% (1/651), and Sarcoptes scabiei 5.2% (34/651). The predilection sites were in the face, ear, and leg areas, and in the axillary, dorsal, abdomen, and scrotum regions of the surveyed animals. An evaluation of farmers' attitudes to ectoparasites was performed using a questionnaire. The findings of this study imply that animals in the investigated area are highly exposed to ectoparasite infestations. Given the importance of ectoparasites in both livestock and human communities, specifically in the health domain, more research into appropriate control strategies is necessary.

Detection of Trypanosoma lewisi from rodents residing in the densely populated residential regions along the coastal areas of Banyuwangi Sub District, Indonesia.

Background: Extensive attention has been devoted to studies of Trypanosoma lewisi in rodents ever since it became recognised as a zoonotic pathogen known as atypical human trypanosomiasis. Regrettably, although T. lewisi infections of small mammals remain significant public health concerns for humans, there is a lack of comprehensive study in Indonesia. Aim: The aim of the study was to detect T. lewisi from rodents residing in the densely populated residential regions along the coastal areas of Banyuwangi Sub District. Methods: A total of 169 rodents were captured across three villages of Kampung Mandar, Lateng and Kepatihan, using rat single live traps. After being euthanized and identified, the blood samples were collected from each rodent via cardiac puncture. Subsequently, the samples were subjected to native (direct blood microscopic examination), microscopic blood smear examination, and molecular analyses utilizing TRYP1S-TRYP1R (623 bp) and LEW1S-LEW1R (220 bp). Results: The results demonstrated that two species of rodents were successfully captured: Rattus norvegicus (65.68%) and Rattus tanezumi (34.32%). Based on the native and microscopic blood smear examinations, the prevalence of T. lewisi across three villages was 23.08% and 24.26% for molecular analysis employing both primers, respectively. The highest prevalence was found in Kampung Mandar Village (31.18%), followed by Kepatihan (16.67%) and Lateng Villages (15.71%). Conclusion: Statistical analysis revealed that T. lewisi was more prevalent in R. tanezumi compared to R. norvegicus. In terms of sex, no statistically significant distinction was observed between female and male infected rodents of either species (p > 0.05), indicating both species can serve as a source of T. lewisi for humans in the surveyed villages.

Seroprevalence of peste des petits ruminants disease in Indonesian buffaloes may be an emerging threat to small ruminants.

Background and Aim: The peste des petit ruminants (PPR) is a disaster-class virus that causes catastrophic drawbacks to small ruminant industries in affected countries. As PPR disease has been reported in neighboring countries, Indonesia, which has a large population of sheep and goats, has become prone to the emerging threat of infection. Because the virus can also infect other animals with subclinical manifestations, large ruminants, such as buffaloes, may play an important role in spreading the virus in the environment. Therefore, the main objective of this study was to identify PPR seroprevalence in the buffalo population of Indonesia. Materials and Methods: A competitive enzyme-linked immunosorbent assay was performed to identify the specific antibody for PPR viruses in the buffalo population using serum bank collection from the National Research and Innovation Agency, Indonesia. Results: PPR virus seroprevalence was detected in buffalo from Central Java, East Java, and East Nusa Tenggara Province in Indonesia. Although seroprevalence was low in the population, the antibody titer was relatively high in the positive samples. Sex and age were identified as determinant factors in the seroprevalence distribution of the buffalo population. Conclusion: The presence of antibodies against the PPR virus in buffaloes may indicate that PPR virus is circulating in the buffalo population of Indonesia.

Anticoccidial activities of Piper betle L essential oil on Eimeria tenella oocysts.

Coccidiosis poses a significant threat to the poultry industry, with synthetic antibiotics and disinfectants being the primary tools for control. This study investigated the potential of Piper betle L essential oil (PBEO) as a natural alternative against Eimeria tenella, one of the most pathogenic Eimeria species affecting poultry. Our findings revealed that PBEO exhibits significant anticoccidial effects through two primary mechanisms: (i) oocysticidal activity by disintegrating oocyst walls and (ii) inhibition of the sporulation process. PBEO demonstrated oocysticidal activities ranging from 8.67 to 95.33% across concentrations from 0.04 to 40%. Notably, at 72 h post-incubation, a 0.04% PBEO concentration significantly reduced the number of sporulated oocysts (P ≤ 0.05) to 71.67%, showing effects comparable to those of formalin. PBEO reduced 50% of oocyst sporulation (IC50) in the concentration of 1.31% at 72 h. Gas chromatography-mass spectrometry (GC-MS) identified the primary constituents of PBEO, including eugenol, beta-caryophyllene, and other key compounds, collectively constituting 96% of the oil. This research underscores the potential of PBEO as a natural anticoccidial agent and lays the groundwork for further studies aimed at identifying, isolating, and developing active compounds that may specifically target the sporogony process in coccidian parasites.

Molecular detection of toxoplasmosis in wild rats using loop-mediated isothermal amplification assay.

Background and Aim: Toxoplasmosis is caused by the parasite Toxoplasma gondii. Cats are the only known hosts that excrete resistant oocysts. Wild rats serve as crucial reservoirs and intermediate hosts for T. gondii's survival and dissemination. Consuming soil and water containing oocysts can lead to illness. This study aimed to estimate the prevalence of toxoplasmosis in wild rats through molecular detection as an indicator of environmental contamination in Surabaya. Materials and Methods: One hundred rats were collected from the three areas (housing, dense settlements, and traditional markets) and distributed into the five zones: West, East, Central, North, and South of Surabaya. Brain tissue samples were extracted using a Geneaid™ (New Taipei City, Taiwan) DNA isolation kit and analyzed through the loop-mediated isothermal amplification (LAMP) method. Results: The study analyzed brain tissue from 100 wild rats, consisting of 77 Rattus tanezumi and 33 Rattus norvegicus, displaying 30% LAMP positivity. The study revealed that 30% (30/100) of wild rats tested were infected with T. gondii. The molecular prevalence rate in male rats was 32.35% (22/68), compared to females with 25% (8/32). 41.9% of the housing population, 33.3% of traditional markets, and 22.6% of dense settlements had the highest molecular prevalence. The high positive molecular rate at the trapping site can be attributed to cats and dense populations. Conclusion: Thirty percentage wild rats were tested positive for toxoplasmosis in Surabaya, East Java, Indonesia using LAMP method. Implementing strict control and monitoring is crucial in preventing the transmission of diseases from wild rats to humans. It is necessary to carry out further research related to genetic analysis of T. gondii to determine the type of T. gondii that infects animals and humans in Surabaya through bioassay and molecular test.

Prevalence of Amphistomes and Fasciola in large ruminants reared by smallholders in Lampung and Banten Provinces, Indonesia.

Background and Aim: Parasitic diseases, including trematode invasions, result in losses to livestock in Indonesia, hindering the government's efforts to achieve meat self-sufficiency. This study aimed to estimate the prevalence of Amphistomes and Fasciola in large ruminants reared by smallholder farmers. Materials and Methods: Fecal samples from 199 buffalo and cattle were collected from the districts of East Lampung (Lampung Province) and Lebak (Banten Province). Fecal samples were examined for the presence of trematode eggs using a sedimentation technique. Results: Parasite invasion rate was 48.2% (95% confidence interval [CI]: 41.3%-55.2%). Rate of invasion was 63.3% (95% CI: 52.7%-73.9%) in Lampung and 38.3% (95% CI: 29.6%-47.0%) in Lebak-Banten. The prevalence of multiple invasions of both Amphistomes and Fasciola was 20% in buffalo and local cattle, whereas invasion rate was 12.8% in crossbred cattle. Invasion rate of Amphistomes alone was 27.1%, and that of Fasciola was 4.5%. A higher invasion rate of Amphistomes (29.8%) occurred in crossbred animals. There were no significant differences between age groups for trematode invasion. The Chi-square test showed that the prevalence of trematode invasion in females was significantly higher than in males (51.5% and 30.0%, respectively). Amphistomes more commonly infected females than males (29.0% and 16.7%, respectively). Conclusion: All breeds were vulnerable to invasion by both trematode species and single invasions with different invasion rates. These findings contribute to determining the magnitude of the disease and provide a basis for studies on prevention and treatment of trematode invasion.

High prevalence of liver fluke infestation, Fasciola gigantica, among slaughtered cattle in Boyolali District, Central Java.

Background: Fasciolosis is a foodborne disease caused by Fasciola sp. infecting ruminants, especially cattle. Fasciolosis remains a significant concern for Veterinary Public Health because of its zoonosis risk and transmission mode. Aim: This study aimed to determine the prevalence and risk factors associated with Fasciola gigantica infestation in cattle at Ampel abbatoir, Central Java, Indonesia. Methods: A cross-sectional study was performed on 585 cattle from February to August 2022. Visual observation postmortem was used to assess Fasciola infection based on adult flukes in liver parenchyma and ductuli biliferi. Results: The overall prevalence of fasciolosis in Ampel abbatoir is high, reaching 25.12% (147/585). The highest prevalence was observed in the Ongole breed, 42.1% (24/57), female cattle, 38.72% (115/297), body condition score criteria of 2 50% (21/42), cattle aged >3.5 years 46.06% (82/178), and cattle originated from outside of Boyolali district 33.33% (71/213). Conclusion: This study showed a high prevalence of fasciolosis in Ampel abbatoir, as shown in the correlation between the risk factors of breed, sex, body condition score (BCS), origin, and age. Because of the high prevalence of fasciolosis in the abattoirs, it is essential to continue performing epidemiology studies in more expansive areas. The subsequent plans are important to reduce the risk of fasciolosis as a threat to productive cattle husbandry and warrant its transmission to humans as a foodborne-zoonotic disease.

First Autochthonous Report on Cattle Babesia naoakii in Central Java, Indonesia, and Identification of Haemaphysalis bispinosa Ticks in the Investigated Area.

In tropical countries, clinical bovine babesiosis is a tick-borne disease primarily caused by Babesia bovis and Babesia bigemina. Here, we investigated 11 cattle with presumptive diagnosis of clinical babesiosis in Boyolali district, Central Java, Indonesia. The majority of the animals were anemic, as evidenced by lower hematocrit, hemoglobin concentration, and red blood cell counts than the normal ranges. Blood DNA was analyzed by a PCR assay targeting the 18S rRNA-ITS region of babesial origin, and the results confirmed that the cattle were infected with Babesia species. The sequencing and phylogenetic analyses demonstrated that the animals were infected with Babesia naoakii. This is the first report of B. naoakii in Indonesia and of B. naoakii-induced clinical bovine babesiosis outside of Sri Lanka. B. naoakii causes a persistent infection, as indicated by positive PCR results for serial blood samples of the circulatory system taken two weeks after treatment. Consequently, subclinical or newly recovered cattle may serve as potential intermediate hosts and infect ticks as definitive hosts to complete the life cycle. To identify potential tick vectors, we collected ticks from cattle, including 11 animals with clinical babesiosis. Based on the morphology and the mitochondrial cytochrome c oxidase subunit 1 (COX1) of collected ticks, we found that all of the collected ticks were Haemaphysalis bispinosa, identifying this tick species as a potential vector of B. naoakii in Indonesia. In this study, the evaluation of local farmers' awareness and practices regarding tick-borne diseases is presented, as disease prevention is also reliant on the implementation of strategies for vector control. Since livestock activities in Java represent the country's busiest animal trade, thereby the spread of disease to other regions is possible through anthropogenic factors. In conclusion, B. naoakii is a causative pathogen of clinical bovine babesiosis autochthonously occurred in this report and further research on B. naoakii-infection is required in other regions of the country. The prompt treatment of the disease seemed crucial for animal survival, which implies the necessity of early diagnosis and a sensitive detection method.

A review of Fasciolopsis buski distribution and control in Indonesia.

Fasciolopsiasis is a parasitic infection caused by the flatworm Fasciolopsis buski. Since 1982, fasciolopsiasis has been reported in Indonesia's Hulu Sungai Utara (HSU) Regency, South Kalimantan Province. Fasciolopsiasis occurs when contaminated raw or undercooked aquatic plants are consumed. Cercariae of the parasite encyst in a variety of aquatic plants and grow into metacercariae that infect and reproduce in the human intestine. Until now, treatment for F. buski infection in the HSU Regency has been comparatively short, with patients receiving only a single dose of praziquantel, 30 mg/kg body weight, without further observation. A long-term effort through health promotion activities and intensive health education, particularly for elementary school children enrolled in the School Health Program, is ongoing to help prevent fasciolopsiasis from spreading and to improve environmental sanitation. Through 2018, intervention efforts successfully reduced the incidence of F. buski infection. Sustaining surveillance and investigation of fasciolopsiasis, including identification of new cases and community education, is critical for the elimination of the parasite from Indonesia. This review describes the spread of F. buski and its possible impact on public health to understand the critical nature of continuing F. buski surveillance and control efforts.

Phylogenetic characterization of Isospora jaracimrmani oocysts from a veiled chameleon (family Chamaeleonidae; Chamaeleo calyptratus) reared at a zoo in Ishikawa, Japan.

Oocysts of Isospora sp. were detected in the feces of a veiled chameleon (family Chamaeleonidae; Chamaeleo calyptratus) kept at a zoo in Ishikawa, Japan. Phylogenetic analysis placed the sequence in the cluster of Isospora spp. isolated from reptiles. Based on a comparison of morphological data of ten previously reported Isospora species from the Chamaeleonidae family, this isolate was morphologically similar to I. jaracimrmani, which has been considered to be a virulent species. This case study suggests the possibility that species of Isospora might not always cause disease because the animal that shed these oocysts showed no symptoms for more than two months.

Prevalence and risk factors associated with Eimeria species infection in cattle of different geographical regions of Indonesia.

BACKGROUND AND AIM: Eimeria spp. are gastrointestinal protozoans that affect animal productivity, thereby causing symptoms that range from bloody diarrhea to death. These symptoms cause economic losses to farmers. The distribution of Eimeria spp. in cattle has, therefore, been reported to have spread widely, especially in the tropics and subtropics. Indonesia is a tropical country at high risk of Eimeria infections. This study aimed to identify the prevalence and risk factors related to the levels of eimeriosis in beef cattle originating from different geographic areas in Indonesia. MATERIALS AND METHODS: Here, 817 fecal samples were collected from beef cattle in Indonesia, including 282 calves, 535 adults, 530 males, and 287 females. In addition, 156 semi-intensively and 661 intensively managed cattle were randomly collected. Then, fecal samples were analyzed by parasitology examinations. RESULTS: Screening examination using the sugar flotation modification method showed that Eimeria spp. were prevalent in Indonesia, as 65.4% of the bacterial strain was detected. The prevalence of identified Eimeria spp. in Indonesia was highest in North Maluku (Maluku Island) (94.1%), whereas the lowest levels were observed in West Java (24.0%) (Java Island). The prevalence was also found to be higher in males (79.3%) than females (51.9%). Similarly, levels in semi-intensively managed cattle (66.7%) were higher than those subjected to intensive management (65.9%). However, its prevalence in calf and adult cattle was similar. CONCLUSION: Bovine eimeriosis spp. were detected at high prevalence in Indonesia, and high-level risks were observed in infected males, including those under the semi-intensive management. In addition, although the results from oocyst examinations were based on qualitative analysis, the endemicity levels of Eimeria spp. among farms in Indonesia should be considered because Eimeria spp. were distributed in most parts of Indonesia. Based on the results of this study, we provide the first information about the prevalence of bovine eimeriosis from different geographical locations in Indonesia, which have differing climates associated with the level of the existing risk factors. Hence, farmers are advised to pay more attention to strict biosecurity techniques on their farms, thereby favoring the early control of bovine eimeriosis.

Detections of gastrointestinal parasites, including Giardia intestinalis and Cryptosporidium spp., in cattle of Banten province, Indonesia.

Gastrointestinal parasites can induce low productivity in livestock by causing acute or chronic enteritis. Veterinarians make great efforts to design rational and effective hygienic protocols for both the prevention and treatment of diarrhea. Although prevalences can vary depending on the examined areas or the ages of the hosts, and the methods used for detections, it is helpful to accumulate data across many areas to evaluate parasitic distribution. A coprological survey in cattle was conducted in Tangerang, Banten Province of Indonesia, in order to determine the prevalence of the parasites, including those of diarrhea-associated diseases. Furthermore, the risk of transmission of Giardia intestinalis and Cryptosporidium spp. to human was genetically analyzed. Gastrointestinal parasites were detected in 87 of 109 cattle samples, including 85 carrying Eimeria spp., 36 carrying Fasciola gigantica, 35 carrying Strongyloides spp., 33 carrying Paramphistomum spp., and 15 carrying Capillaria spp. Giardia intestinalis and Cryptosporidium spp., parasites with zoonotic potential, were detected in 9 and 1 cattle samples, respectively. Molecular analyses identified the G. intestinalis isolate as a member of Assemblage E, which has been recently detected in humans in another country. These results may be helpful in understanding the hygienic risk affecting the livestock productivity and zoonotic potential of cattle in Indonesia.

Molecular characterization of highly pathogenic Eimeria species among beef cattle on Java Island, Indonesia.

Gastrointestinal parasites including Eimeria spp. are known to affect domestic animal productivity causing watery or lethal bloody diarrhea. However, there are few reports on the detailed distribution of bovine Eimeria spp. in cattle, particularly in developing tropical and sub-tropical areas. Using a total of 289 fecal samples collected from beef cattle on Java Island, one of the five main islands of Indonesia, fecal examinations by the Whitlock and sugar flotation methods and molecular surveys were conducted to reveal the prevalence of 6 Eimeria spp. As a result of morphological screening using Whitlock methods and sugar flotation, Eimeria spp. prevalences of 9.4% and 52.3% were confirmed, respectively. The prevalence was higher in younger cattle [under 1 year (63.9%), 1-2 years (75.0%) and more than in 2 year old cattle (42.3%)]. The prevalences of identified species were as follows: 10.4% for E. bovis, 2.8% for E. ellipsoidalis, 2.1% for E. alabamensis, 1.4% for E. zuernii, 1.1% for E. auburnensis, and 0.4% for E. cylindrica. Moreover, prevalences of 12.8% for Strongyloides papillosus, 7.3% for Trichuris globulosa, and 0.3% for Capillaria bovis were detected. Although the average number of oocysts per gram of feces was <100 among the positive samples, and cases of heavy infection were limited, the endemicity of these pathogenic Eimeria species among farms in Indonesia should be noted.

Chicken Coccidiosis in Central Java, Indonesia: A Recent Update.

Avian coccidiosis is a huge problem worldwide. Heavily infected animals that show severe clinical signs and coccidiostat resistance are causing important economic losses. The present study aimed to update the recent cases of coccidiosis in Central Java, Indonesia, and to show the importance of the disease in the region. A total of 699 samples were obtained from different chicken breed. Different Eimeria species were detected in 175 individuals (25.04%). Three different groups of chicken breed were considered: local chicken (autochthonous chickens of Sentul and Jawa), commercial broiler, and layer. Broiler chickens showed the highest prevalence of infection (34%), followed by layer (26.26%) and local chickens (10.45%). Mild to severe clinical signs of avian coccidiosis were observed in 42% of the infected animals, while 58% of the infected animals showed no clinical signs other than low feed conversion rates. Seven different Eimeria species were identified: E. tenella was the most prevalent (43.3%), followed by E. maxima (26.3%), E. necatrix (15.7%), E. acervulina (8%), E. praecox (3.1%), E. mitis (2.2%), and E. brunetti (1.3%). Coinfections with several Eimeria species were diagnosed. With this study we found massive usage of coccidiostat in the region even though its usage cannot guarantee coccidiosis-free chicken production.

Integrative taxonomy to investigate species boundaries within Culicoides (Diptera: Ceratopogonidae): a case study using subgenus Avaritia from Australasia and Eastern Asia.

In this study, species boundaries were examined for 15 described and 2 undescribed species within the economically important Culicoides subg. Avaritia Fox from Australasia and Eastern Asia. We used an integrative taxonomic approach incorporating DNA barcoding, nuclear gene sequencing, and retrospective morphological analyses. Some arbovirus vector species such as Culicoides fulvus Sen and Das Gupta and Culicoides wadai Kitaoka were genetically and morphologically uniform across sampled distributions, but others including Culicoides actoni Smith and Culicoides brevipalpis Delfinado contained 2 or more genetically independent populations of 'cryptic species' that in some cases were sympatric. Some of these 'cryptic species' exhibited consistent morphological differences, while differences are yet to be found for others species. Additionally, an undescribed species, C. Avaritia sp. No. 3, was found to be synonymous with C. fulvus. These results refine our understanding of the distribution of individual species of C. subg. Avaritia and demonstrate that species descriptions and distribution records need revision for part of the Culicoides fauna. Furthermore, because vector competence studies for most of these species are based entirely on Australian populations, the competence of the putative cryptic species identified elsewhere will require independent assessment. Finally, integrative taxonomic assessment requires genetic and morphological assessment of material from the type localities in order to clarify the status and distribution of species, especially for clades containing cryptic species. International collaboration is needed to facilitate this research.