Standardizing Visual Control Devices for Tsetse Flies: East African Species Glossina fuscipes fuscipes and Glossina tachinoides

BackgroundRiverine species of tsetse are responsible for most human African trypanosomiasis (HAT) transmission and are also important vectors of animal trypanosomiasis. This study concerns the development of visual control devices for two such species, Glossina fuscipes fuscipes and Glossina tachinoides, at the eastern limits of their continental range. The goal was to determine the most long-lasting, practical and cost-effective visually attractive device that induces the strongest landing responses in these species for use as insecticide-impregnated tools in vector population suppression.Conclusions/SignificanceTaking into account practical considerations and fly preferences for edges and colours, we propose a 0.5×0.75 m blue-black target as a simple cost-effective device for management of G. f. fuscipes and G. tachinoides, impregnated with insecticide for control and covered with adhesive film for population sampling.     

Standardising Visual Control Devices for Tsetse Flies: Central and West African Species Glossina palpalis palpalis

Background

Glossina palpalis palpalis (G. p. palpalis) is one of the principal vectors of sleeping sickness and nagana in Africa with a geographical range stretching from Liberia in West Africa to Angola in Central Africa. It inhabits tropical rain forest but has also adapted to urban settlements. We set out to standardize a long-lasting, practical and cost-effective visually attractive device that would induce the strongest landing response by G. p. palpalis for future use as an insecticide-impregnated tool in area-wide population suppression of this fly across its range.

Methodology/Principal Findings

Trials were conducted in wet and dry seasons in the Ivory Coast, Cameroon, the Democratic Republic of Congo and Angola to measure the performance of traps (biconical, monoconical and pyramidal) and targets of different sizes and colours, with and without chemical baits, at different population densities and under different environmental conditions. Adhesive film was used as a practical enumerator at these remote locations to compare landing efficiencies of devices. Independent of season and country, both phthalogen blue-black and blue-black-blue 1 m² targets covered with adhesive film proved to be as good as traps in phthalogen blue or turquoise blue for capturing G. p. palpalis. Trap efficiency varied (8–51%). There was no difference between the performance of blue-black and blue-black-blue 1 m² targets. Baiting with chemicals augmented the overall performance of targets relative to traps. Landings on smaller phthalogen blue-black 0.25 m² square targets were not significantly different from either 1 m² blue-black-blue or blue-black square targets. Three times more flies were captured per unit area on the smaller device.

Conclusions/Significance

Blue-black 0.25 m² cloth targets show promise as simple cost effective devices for management of G. p. palpalis as they can be used for both control when impregnated with insecticide and for population sampling when covered with adhesive film.     

Prospects for the Development of Odour Baits to Control the Tsetse Flies Glossina tachinoides and G. palpalis s.l.

Field studies were done of the responses of Glossina palpalis palpalis in Côte d''Ivoire, and G. p. gambiensis and G. tachinoides in Burkina Faso, to odours from humans, cattle and pigs. Responses were measured either by baiting (1.) biconical traps or (2.) electrocuting black targets with natural host odours. The catch of G. tachinoides from traps was significantly enhanced (∼5×) by odour from cattle but not humans. In contrast, catches from electric targets showed inconsistent results. For G. p. gambiensis both human and cattle odour increased (>2×) the trap catch significantly but not the catch from electric targets. For G. p. palpalis, odours from pigs and humans increased (∼5×) the numbers of tsetse attracted to the vicinity of the odour source but had little effect on landing or trap-entry. For G. tachinoides a blend of POCA (P = 3-n-propylphenol; O = 1-octen-3-ol; C = 4-methylphenol; A = acetone) alone or synthetic cattle odour (acetone, 1-octen-3-ol, 4-methylphenol and 3-n-propylphenol with carbon dioxide) consistently caught more tsetse than natural cattle odour. For G. p. gambiensis, POCA consistently increased catches from both traps and targets. For G. p. palpalis, doses of carbon dioxide similar to those produced by a host resulted in similar increases in attraction. Baiting traps with super-normal (∼500 mg/h) doses of acetone also consistently produced significant but slight (∼1.6×) increases in catches of male flies. The results suggest that odour-baited traps and insecticide-treated targets could assist the AU-Pan African Tsetse and Trypanosomiasis Eradication Campaign (PATTEC) in its current efforts to monitor and control Palpalis group tsetse in West Africa. For all three species, only ∼50% of the flies attracted to the vicinity of the trap were actually caught by it, suggesting that better traps might be developed by an analysis of the visual responses and identification of any semiochemicals involved in short-range interaction.     

Copulatory Dialogues Between Male and Female Tsetse Flies (Diptera: Muscidae: Glossina pallidipes)

Traditional views of copulation and sperm transfer supposed that females are passive participants. Recent discoveries suggest, however, that females actively influence the chances that a copulation will result in fertilization of their eggs, and that they sometimes signal to males during copulation in order to elicit male responses. This paper concerns two apparent female signaling behaviors, wing vibration and body shaking, in the tsetse fly Glossina pallidipes, a species in which the male squeezes the female’s abdomen rhythmically with his powerful genitalia. Vibration was associated with male squeezes at several levels of analysis. Its coordination with male behavior suggests that vibration functions as a signal that induces the male to interrupt squeezes, but that does not forcefully dislodge the male. The female tended to vibrate her wings soon after the male began a squeeze; and when the female vibrated her wings during a squeeze, the squeeze tended to be shorter. Female body shaking was usually elicited by especially powerful squeezes. Previous studies showed that stimuli from male structures that squeeze the female probably function to induce her to ovulate, to facilitate movement of sperm into her spermathecae, and to reject the sexual advances of additional males. This study is one of the first to document an exchange of signals between male and female insects during copulation, and extends the new field of research on copulatory dialogues.     

Laboratory Colonisation and Genetic Bottlenecks in the Tsetse Fly Glossina pallidipes

Background

The IAEA colony is the only one available for mass rearing of Glossina pallidipes, a vector of human and animal African trypanosomiasis in eastern Africa. This colony is the source for Sterile Insect Technique (SIT) programs in East Africa. The source population of this colony is unclear and its genetic diversity has not previously been evaluated and compared to field populations.

Methodology/Principal Findings

We examined the genetic variation within and between the IAEA colony and its potential source populations in north Zimbabwe and the Kenya/Uganda border at 9 microsatellites loci to retrace the demographic history of the IAEA colony. We performed classical population genetics analyses and also combined historical and genetic data in a quantitative analysis using Approximate Bayesian Computation (ABC). There is no evidence of introgression from the north Zimbabwean population into the IAEA colony. Moreover, the ABC analyses revealed that the foundation and establishment of the colony was associated with a genetic bottleneck that has resulted in a loss of 35.7% of alleles and 54% of expected heterozygosity compared to its source population. Also, we show that tsetse control carried out in the 1990''s is likely reduced the effective population size of the Kenya/Uganda border population.

Conclusions/Significance

All the analyses indicate that the area of origin of the IAEA colony is the Kenya/Uganda border and that a genetic bottleneck was associated with the foundation and establishment of the colony. Genetic diversity associated with traits that are important for SIT may potentially have been lost during this genetic bottleneck which could lead to a suboptimal competitiveness of the colony males in the field. The genetic diversity of the colony is lower than that of field populations and so, studies using colony flies should be interpreted with caution when drawing general conclusions about G. pallidipes biology.     

Odorant and Gustatory Receptors in the Tsetse Fly Glossina morsitans morsitans

Tsetse flies use olfactory and gustatory responses, through odorant and gustatory receptors (ORs and GRs), to interact with their environment. Glossina morsitans morsitans genome ORs and GRs were annotated using homologs of these genes in Drosophila melanogaster and an ab initio approach based on OR and GR specific motifs in G. m. morsitans gene models coupled to gene ontology (GO). Phylogenetic relationships among the ORs or GRs and the homologs were determined using Maximum Likelihood estimates. Relative expression levels among the G. m. morsitans ORs or GRs were established using RNA-seq data derived from adult female fly. Overall, 46 and 14 putative G. m. morsitans ORs and GRs respectively were recovered. These were reduced by 12 and 59 ORs and GRs respectively compared to D. melanogaster. Six of the ORs were homologous to a single D. melanogaster OR (DmOr67d) associated with mating deterrence in females. Sweet taste GRs, present in all the other Diptera, were not recovered in G. m. morsitans. The GRs associated with detection of CO₂ were conserved in G. m. morsitans relative to D. melanogaster. RNA-sequence data analysis revealed expression of GmmOR15 locus represented over 90% of expression profiles for the ORs. The G. m. morsitans ORs or GRs were phylogenetically closer to those in D. melanogaster than to other insects assessed. We found the chemoreceptor repertoire in G. m. morsitans smaller than other Diptera, and we postulate that this may be related to the restricted diet of blood-meal for both sexes of tsetse flies. However, the clade of some specific receptors has been expanded, indicative of their potential importance in chemoreception in the tsetse.     

Phylogeography and Population Structure of Glossina fuscipes fuscipes in Uganda: Implications for Control of Tsetse

Background

Glossina fuscipes fuscipes, a riverine species of tsetse, is the main vector of both human and animal trypanosomiasis in Uganda. Successful implementation of vector control will require establishing an appropriate geographical scale for these activities. Population genetics can help to resolve this issue by characterizing the extent of linkage among apparently isolated groups of tsetse.

Methodology/Principal Findings

We conducted genetic analyses on mitochondrial and microsatellite data accumulated from approximately 1000 individual tsetse captured in Uganda and neighboring regions of Kenya and Sudan. Phylogeographic analyses suggested that the largest scale genetic structure in G. f. fuscipes arose from an historical event that divided two divergent mitochondrial lineages. These lineages are currently partitioned to northern and southern Uganda and co-occur only in a narrow zone of contact extending across central Uganda. Bayesian assignment tests, which provided evidence for admixture between northern and southern flies at the zone of contact and evidence for northerly gene flow across the zone of contact, indicated that this structure may be impermanent. On the other hand, microsatellite structure within the southern lineage indicated that gene flow is currently limited between populations in western and southeastern Uganda. Within regions, the average F_ST between populations separated by less than 100 km was less than ∼0.1. Significant tests of isolation by distance suggested that gene flow is ongoing between neighboring populations and that island populations are not uniformly more isolated than mainland populations.

Conclusions/Significance

Despite the presence of population structure arising from historical colonization events, our results have revealed strong signals of current gene flow within regions that should be accounted for when planning tsetse control in Uganda. Populations in southeastern Uganda appeared to receive little gene flow from populations in western or northern Uganda, supporting the feasibility of area wide control in the Lake Victoria region by the Pan African Tsetse and Trypanosomiasis Eradication Campaign.     

A Low-cost Contamination Device for Infecting Adult Tsetse Flies, Glossina spp., with the Entomopathogenic Fungus Metarhizium anisopliae in the Field

A low-cost device for infecting adult tsetse fly, Glossina fuscipes fuscipes , with the entomopathogenic fungus Metarhizium anisopliae was designed and tested in the field. Tsetse flies that are attracted to the trap entered the contamination device and ultimately became infected with the fungus. Traps exposed to the sun attracted more flies than did the ones placed in the shade. The time spent by single flies in the contamination device varied between 5- 189 s, and the subsequent number of conidia collected varied between 1.6 ×10 5 conidia and 40.5 ×10 5 conidia per fly, and largely depended on the behavior of individual flies. Dry conidia of M. anisopliae in the device retained their viability for 31 days in the field, and efficacy against G. fuscipes was not affected.     

Optimal Strategies for Controlling Riverine Tsetse Flies Using Targets: A Modelling Study

BackgroundTsetse flies occur in much of sub-Saharan Africa where they transmit the trypanosomes that cause the diseases of sleeping sickness in humans and nagana in livestock. One of the most economical and effective methods of tsetse control is the use of insecticide-treated screens, called targets, that simulate hosts. Targets have been ~1m², but recently it was shown that those tsetse that occupy riverine situations, and which are the main vectors of sleeping sickness, respond well to targets only ~0.06m². The cheapness of these tiny targets suggests the need to reconsider what intensity and duration of target deployments comprise the most cost-effective strategy in various riverine habitats.Conclusion/SignificanceSeasonal use of tiny targets deserves field trials. The ability to recognise habitat that contains tsetse populations which are not self-sustaining could improve the planning of all methods of tsetse control, against any species, in riverine, savannah or forest situations. Criteria to assist such recognition are suggested.     

Rickettsia Species in African Anopheles Mosquitoes

Background

There is higher rate of R. felis infection among febrile patients than in healthy people in Sub-Saharan Africa, predominantly in the rainy season. Mosquitoes possess a high vectorial capacity and, because of their abundance and aggressiveness, likely play a role in rickettsial epidemiology.

Methodology/Principal Findings

Quantitative and traditional PCR assays specific for Rickettsia genes detected rickettsial DNA in 13 of 848 (1.5%) Anopheles mosquitoes collected from Côte d’Ivoire, Gabon, and Senegal. R. felis was detected in one An. gambiae molecular form S mosquito collected from Kahin, Côte d’Ivoire (1/77, 1.3%). Additionally, a new Rickettsia genotype was detected in five An. gambiae molecular form S mosquitoes collected from Côte d’Ivoire (5/77, 6.5%) and one mosquito from Libreville, Gabon (1/88, 1.1%), as well as six An. melas (6/67, 9%) mosquitoes collected from Port Gentil, Gabon. A sequence analysis of the gltA, ompB, ompA and sca4 genes indicated that this new Rickettsia sp. is closely related to R. felis. No rickettsial DNA was detected from An. funestus, An. arabiensis, or An. gambiae molecular form M mosquitoes. Additionally, a BLAST analysis of the gltA sequence from the new Rickettsia sp. resulted in a 99.71% sequence similarity to a species ({"type":"entrez-nucleotide","attrs":{"text":"JQ674485","term_id":"386870496","term_text":"JQ674485"}}JQ674485) previously detected in a blood sample of a Senegalese patient with a fever from the Bandafassi village, Kedougou region.

Conclusion

R. felis was detected for the first time in An. gambiae molecular form S, which represents the major African malaria vector. The discovery of R. felis, as well as a new Rickettsia species, in mosquitoes raises new issues with respect to African rickettsial epidemiology that need to be investigated, such as bacterial isolation, the degree of the vectorial capacity of mosquitoes, the animal reservoirs, and human pathogenicity.     

Tsetse Control and Gambian Sleeping Sickness; Implications for Control Strategy

Background

Gambian sleeping sickness (human African trypanosomiasis, HAT) outbreaks are brought under control by case detection and treatment although it is recognised that this typically only reaches about 75% of the population. Vector control is capable of completely interrupting HAT transmission but is not used because it is considered too expensive and difficult to organise in resource-poor settings. We conducted a full scale field trial of a refined vector control technology to determine its utility in control of Gambian HAT.

Methods and Findings

The major vector of Gambian HAT is the tsetse fly Glossina fuscipes which lives in the humid zone immediately adjacent to water bodies. From a series of preliminary trials we determined the number of tiny targets required to reduce G. fuscipes populations by more than 90%. Using these data for model calibration we predicted we needed a target density of 20 per linear km of river in riverine savannah to achieve >90% tsetse control. We then carried out a full scale, 500 km² field trial covering two HAT foci in Northern Uganda to determine the efficacy of tiny targets (overall target density 5.7/km²). In 12 months, tsetse populations declined by more than 90%. As a guide we used a published HAT transmission model and calculated that a 72% reduction in tsetse population is required to stop transmission in those settings.

Interpretation

The Ugandan census suggests population density in the HAT foci is approximately 500 per km². The estimated cost for a single round of active case detection (excluding treatment), covering 80% of the population, is US$433,333 (WHO figures). One year of vector control organised within the country, which can completely stop HAT transmission, would cost US$42,700. The case for adding this method of vector control to case detection and treatment is strong. We outline how such a component could be organised.     

Standardizing visual control devices for tsetse flies: West African species Glossina tachinoides, G. palpalis gambiensis and G. morsitans submorsitans

Here we describe field trials designed to standardize tools for the control of Glossina tachinoides, G. palpalis gambiensis and G.morsitans submorsitans in West Africa based on existing trap/target/bait technology. Blue and black biconical and monoconical traps and 1 m(2) targets were made in either phthalogen blue cotton, phthalogen blue cotton/polyester or turquoise blue polyester/viscose (all with a peak reflectance between 450-480 nm) and a black polyester. Because targets were covered in adhesive film, they proved to be significantly better trapping devices than either of the two trap types for all three species (up to 14 times more for G. tachinoides, 10 times more for G. palpalis gambiensis, and 6.5 times for G. morsitans submorsitans). The relative performance of the devices in the three blue cloths tested was the same when unbaited or baited with a mixture of phenols, 1-octen-3-ol and acetone. Since insecticide-impregnated devices act via contact with flies, we enumerated which device (traps or targets) served as the best object for flies to land on by also covering the cloth parts of traps with adhesive film. Despite the fact that the biconical trap proved to be the best landing device for the three species, the difference over the target (20-30%) was not significant. This experiment also allowed an estimation of trap efficiency, i.e. the proportion of flies landing on a trap that are caught in its cage. A low overall efficiency of the biconical or monoconical traps of between 11-24% was recorded for all three species. These results show that targets can be used as practical devices for population suppression of the three species studied. Biconical traps can be used for population monitoring, but a correction factor of 5-10 fold needs to be applied to captures to compensate for the poor trapping efficiency of this device for the three species.     

Towards an Early Warning System for Rhodesian Sleeping Sickness in Savannah Areas: Man-Like Traps for Tsetse Flies

Background

In the savannahs of East and Southern Africa, tsetse flies (Glossina spp.) transmit Trypanosoma brucei rhodesiense which causes Rhodesian sleeping sickness, the zoonotic form of human African trypanosomiasis. The flies feed mainly on wild and domestic animals and are usually repelled by humans. However, this innate aversion to humans can be undermined by environmental stresses on tsetse populations, so increasing disease risk. To monitor changes in risk, we need traps designed specifically to quantify the responsiveness of savannah tsetse to humans, but the traps currently available are designed to simulate other hosts.

Methodology/Principal Findings

In Zimbabwe, two approaches were made towards developing a man-like trap for savannah tsetse: either modifying an ox-like trap or creating new designs. Tsetse catches from a standard ox-like trap used with and without artificial ox odor were reduced by two men standing nearby, by an average of 34% for Glossina morsitans morsitans and 56% for G. pallidipes, thus giving catches more like those made by hand-nets from men. Sampling by electrocuting devices suggested that the men stopped flies arriving near the trap and discouraged trap-entering responses. Most of human repellence was olfactory, as evidenced by the reduction in catches when the trap was used with the odor of hidden men. Geranyl acetone, known to occur in human odor, and dispensed at 0.2 mg/h, was about as repellent as human odor but not as powerfully repellent as wood smoke. New traps looking and smelling like men gave catches like those from men.

Conclusion/Significance

Catches from the completely new man-like traps seem too small to give reliable indices of human repellence. Better indications would be provided by comparing the catches of an ox-like trap either with or without artificial human odor. The chemistry and practical applications of the repellence of human odor and smoke deserve further study.     

The distribution of repetitive DNAs between regular and supernumerary chromosomes in Species of Glossina (Tsetse): a two-step process in the origin of supernumeraries

Several species of tsetse fly within the Morsitans and Fusca subgenera of Glossina contain supernumerary (B) chromosomes. Previous studies on the meiotic behaviour of chromosomes (Southern and Pell, 1973) and the C-band patterns (Jordan et al., 1977) have indicated a close similarity between the Y chromosome and the supernumeraries. The distributions of the highly abundant families of DNA (satellite DNAs) between the autosomes, sex chromosomes and B chromosomes of G.m. morsitans, G. austeni and G. pallidipes have been examined by in situ hybridisation. In addition, the organisation and sequence homologies of satellite DNAs have been examined by restriction enzymes and heterologous hybridisations in in situ and Southern transfer conditions. The majority of satellite sequences that are homologous between species are distributed in several different arrangements between A and B chromosome telomeres with minority sequences at some centromeric and intercalary locations. There is no extensive satellite DNA similarity between the Y and B chromosomes. We suggest that the Y and B chromosome associations and synchronous allocycly during meiosis are the result of extensive heterochromatinisation of these two chromosome types, that is probably a reflection of two separate stages involved in the generation of the B chromosomes in the genus. The independent evolution of satellites and supernumeraries is discussed.     

Mermithid Parasitism of Black Flies (Diptera: Simuliidae)

Mermithid nematodes are common parasites of black flies and play a significant role in the natural regulation of these medically important insects. Infection levels tend to he moderate and perennial, with epizootics rare and highly localized. Mermithid parasitism almost invariably results in the death of the black fly, and thus considerable attention has focused on the potential of these nematodes as biocontrol agents. Early instar black fly larvae appear most susceptible to infection, and integumental penetration hy mermithid preparasites is the only known mode of entry. Postparasitic nematodes typically emerge before host pupation. However, carryover of parasitism into adult simuliids is an important mechanism for local dispersal and recolonization of upstream areas. Following emergence, the mermithids molt to the adult stage. Copulation ensues, the females then laying eggs which eventually give rise to the next generation of infective preparasites. The number of described species is conservatively estimated at 35-40, with most species within the genera Mesomermis, Gastromermis, and Isomermis. The taxonomy of this group of mermithids is a challenging and little explored area. Host-specificity statements, therefore, must be made cautiously because of these systematic problems and others within the Simuliidae. In most instances, temporal and spatial factors limit the host range of these mermithids among simuliid species. Differential susceptibilities anmng larvae concurrently present within the same microhabitat probably reflect varying degrees of host attractiveness and behavioral-physiological resistance. Effects of parasitism on the host may include prevention of metamorphosis, sterility, intersexual development, and behavior modification. Evaluation of the technical feasibility of mermithid control of black flies has been stymied by the limitations of current inoculum-production technology. Continued advances in in vivo and in vitro culture methods are required to accelerate the research process.     

Comparison of Two Steinernematid Species for Control of the Root Weevil Diaprepes abbreviatus

Steinernema carpocapsae Weiser All strain was compared to Steinernema riobravis Cabanillas, Poinar, and Raulston for control of the root weevil, Diaprepes abbreviatus (L.), in the laboratory and in potted citrus. In the laboratory bioassay, D. abbreviatus larvae were exposed to 30, 60, and 120 nematodes/cm³ in sand. Insect mortality 1 week after application was greater (P ≤ 0.05) for S. riobravis than for S. carpocapsae in the laboratory bioassay. In the greenhouse bioassay, D. abbreviatus larvae were exposed to 3 and 9 nematodes per cm³ of soil in potted citrus. Again, at each rate, mortality was greater (P ≤ 0.05) in pots treated with S. riobravis than in pots treated with S. carpocapsae. The results of this study suggest that S. riobravis is a better biological control agent against D. abbreviatus larvae in potted plants than S. carpocapsae.     

Prospects for Developing Odour Baits To Control Glossina fuscipes spp., the Major Vector of Human African Trypanosomiasis

We are attempting to develop cost-effective control methods for the important vector of sleeping sickness, Glossina fuscipes spp. Responses of the tsetse flies Glossina fuscipes fuscipes (in Kenya) and G. f. quanzensis (in Democratic Republic of Congo) to natural host odours are reported. Arrangements of electric nets were used to assess the effect of cattle-, human- and pig-odour on (1) the numbers of tsetse attracted to the odour source and (2) the proportion of flies that landed on a black target (1×1 m). In addition responses to monitor lizard (Varanus niloticus) were assessed in Kenya. The effects of all four odours on the proportion of tsetse that entered a biconical trap were also determined. Sources of natural host odour were produced by placing live hosts in a tent or metal hut (volumes≈16 m³) from which the air was exhausted at ∼2000 L/min. Odours from cattle, pigs and humans had no significant effect on attraction of G. f. fuscipes but lizard odour doubled the catch (P<0.05). Similarly, mammalian odours had no significant effect on landing or trap entry whereas lizard odour increased these responses significantly: landing responses increased significantly by 22% for males and 10% for females; the increase in trap efficiency was relatively slight (5–10%) and not always significant. For G. f. quanzensis, only pig odour had a consistent effect, doubling the catch of females attracted to the source and increasing the landing response for females by ∼15%. Dispensing CO₂ at doses equivalent to natural hosts suggested that the response of G. f. fuscipes to lizard odour was not due to CO₂. For G. f. quanzensis, pig odour and CO₂ attracted similar numbers of tsetse, but CO₂ had no material effect on the landing response. The results suggest that identifying kairomones present in lizard odour for G. f. fuscipes and pig odour for G. f. quanzensis may improve the performance of targets for controlling these species.     

Standardizing Immunohistochemistry: A New Reference Control for Detecting Staining Problems

A new standardized immunohistochemistry (IHC) control for breast cancer testing comprises formalin-fixed human epidermal growth factor receptor 2, estrogen receptor, or progesterone receptor peptide antigens covalently attached to 8-µm glass beads. The antigen-coated beads are suspended in a liquid matrix that hardens upon pipetting onto a glass microscope slide. The antigen-coated beads remain in place through deparaffinization, antigen retrieval, and immunostaining. The intensity of the beads’ stain provides feedback regarding the efficacy of both antigen retrieval and immunostaining. As a first report, we tested the sensitivity and specificity of the new IHC controls (“IHControls”). To evaluate sensitivity, various staining problems were simulated. IHControls detected primary and secondary reagent degradation similarly to tissue controls. This first group of IHControls behaved similarly to tissue controls expressing high concentrations of the antigen. The IHControls were also able to detect aberrations in antigen retrieval, as simulated by sub-optimal times or temperatures. Specificity testing revealed that each antigen-coated bead was specific for its cognate IHC test antibody. The data support the conclusion that, like tissue controls, IHControls are capable of verifying the analytic components of an immunohistochemical stain. Unlike tissue controls, IHControls are prepared in large bulk lots, fostering day-to-day reproducibility that can be standardized across laboratories.