Exposure to Zika and chikungunya viruses impacts aspects of the vectorial capacity of Aedes aegypti and Culex quinquefasciatus.

Zika (ZIKV) and chikungunya (CHIKV) are arboviruses that cause infections in humans and can cause clinical complications, representing a worldwide public health problem. Aedes aegypti is the primary vector of these pathogens and Culex quinquefasciatus may be a potential ZIKV vector. This study aimed to evaluate fecundity, fertility, survival, longevity, and blood feeding activity in Ae. aegypti after exposure to ZIKV and CHIKV and, in Cx. quinquefasciatus exposed to ZIKV. Three colonies were evaluated: AeCamp (Ae. aegypti-field), RecL (Ae. aegypti-laboratory) and CqSLab (Cx. quinquefasciatus-laboratory). Seven to 10 days-old females from these colonies were exposed to artificial blood feeding with CHIKV or ZIKV. CHIKV caused reduction in fecundity and fertility in AeCamp and reduction in survival and fertility in RecL. ZIKV impacted survival in RecL, fertility in AeCamp and, fecundity and fertility in CqSLab. Both viruses had no effect on blood feeding activity. These results show that CHIKV produces a higher biological cost in Ae. aegypti, compared to ZIKV, and ZIKV differently alters the biological performance in colonies of Ae. aegypti and Cx. quinquefasciatus. These results provide a better understanding over the processes of virus-vector interaction and can shed light on the complexity of arbovirus transmission.

New weapons against the disease vector Aedes aegypti: From natural products to nanoparticles.

Despite the global burden of viral diseases transmitted by Aedes aegypti, there is a lack of effective means of prevention and treatment. Strategies for vector control include chemical and biological approaches such as organophosphates and Bacillus thuringiensis var. israelensis (Bti), among others. However, important concerns are associated, such as resistance in mosquito larvae and deleterious effects on non-target organisms. In this scenario, novel approaches against A. aegypti have been investigated, including natural products (e.g. vegetable oil and extracts) and nanostructured systems. This review focuses on potential strategies for fighting A. aegypti, highlighting plant-based materials and nanomaterials able to induce toxic effects on egg, larva, pupa and adult mosquitoes. Issues including aspects of conventional vector control strategies are presented, and finally new insights on development of eco-friendly nanoformulations against A. aegypti are discussed.

Mapping Aedes aegypti indoor resting behavior reveals a preference vulnerable to householder-led vector control.

Many mosquito vectors rest inside human habitations, a behavioral trait that is exploited for vector control by indoor residual spraying (IRS) of interior walls with insecticide. Although IRS and its refined version targeted IRS are very effective against Aedes aegypti, they are expensive and logistically challenging to deliver in densely populated urban areas where outbreaks of dengue and other arboviruses are the greatest challenge. In experiments in Recife, Brazil, we set out to quantify the indoor resting behavior of Ae. aegypti at a level beyond that previously reported. We found that significantly more Ae. aegypti males, unfed and fed females visited the base of walls (height 0-20 cm, corresponding to 12.3% of the total wall surface) more frequently than upper wall areas, with the difference more pronounced at higher temperatures. When the lowest 20 cm of the walls was treated with an appropriate insecticide and colored black, we recorded up to 85% cumulative mortality after 24-h exposure in the experimental room. The findings are significant because feasibly, householders could treat this small and accessible target zone manually, without the need for visits by costly IRS teams or equipment, reducing insecticide use and enabling communities to actively protect their own indoor environment.

Double BR-OVT: a new trap model for collecting eggs and adult mosquitoes from Culex quinquefasciatus and Aedes spp.

The circulation of arboviruses throughout the world and the maintenance of lymphatic filariasis endemicity in tropical countries, combined with the lack of vaccines and specific treatments, highlight the importance of reducing the populations of mosquitoes involved in the transmission of these pathogens, Aedes aegypti and Culex quinquefasciatus. To contribute to the development of new strategies for monitoring and controlling these culicids, we evaluated the performance of the Double BR-OVT trap individually and in pairs, in the field. After 18 months, the Double BR-OVT traps captured a mean of 3.5 ± 7.4 and 1.8 ± 3.2 of Culex and Aedes /residence/cycle, respectively, in addition to 410 ± 588.3 Aedes eggs/residence/cycle. When installed in pairs, the Double BR-OVT traps collected three times more adult mosquitoes of C. quinquefasciatus (9.4 ± 8.3 Culex /residence/bimester) and two times more Aedes spp. (3 ± 3.2 Aedes /residence/bimester) in comparison with the traps installed individually (2.6 ± 7.1 and 1.5 ± 3.2 Culex and Aedes /residence/bimester, respectively) (p < 0.05). The Double BR-OVT trap has an exceptional advantage: it aggregates different functionalities into a single instrument, as this type of trap can concomitantly collect eggs and adult mosquitoes of C. quinquefasciatus and A. aegypti , a feature that makes it a potentially useful tool among the strategies for monitoring and controlling these mosquitoes.

Infestation of an endemic arbovirus area by sympatric populations of Aedes aegypti and Aedes albopictus in Brazil.

BACKGROUND Aedes aegypti and Aedes albopictus are the most important arbovirus vectors in the world. OBJECTIVES This study aimed to investigate and compare the infestation pattern of these species in a neighbourhood of Recife, Brazil, endemic for arboviruses in 2005 (T1) and 2013 (T2). METHODS Infestation, distribution and relative abundance of these sympatric species were recorded by egg collection using a network of 59 sentinel ovitraps (s-ovt) at fixed sampling stations for 12 months in T1 and T2. FINDINGS A permanent occupation pattern was detected which was characterised by the presence of egg-laying females of one or both species with a high ovitrap positivity index (94.3 to 100%) throughout both years analysed. In terms of abundance, the total of eggs collected was lower (p < 0.005) in T2 (146,153) than in T1 (281,103), although ovitraps still displayed a high index of positivity. The spatial distribution showed the presence of both species in 65.1% of the 148 s-ovt assessed, while a smaller number of traps exclusively contained Ae. aegypti (22%) or Ae. albopictus (13.2%) eggs. MAIN CONCLUSIONS Our comparative analysis demonstrated the robustness of the spatial occupation and permanence of Ae. aegypti and Ae. albopictus populations in this endemic urban area.

A popular Indian clove-based mosquito repellent is less effective against Culex quinquefasciatus and Aedes aegypti than DEET.

Insect repellents are widely used as the first line of defense against mosquito bites and transmission of disease-causing agents. However, the cost of daily applications of even the most affordable and the gold standard of insect repellents, DEET, is still high for low-income populations where repellents are needed the most. An Indian clove-based homemade recipe has been presented as a panacea. We analyzed this homemade repellent and confirmed by behavioral measurements and odorant receptor responses that eugenol is the active ingredient in this formulation. Prepared as advertised, this homemade repellent is ineffective, whereas 5x more concentrated extracts from the brand most enriched in eugenol showed moderate repellency activity against Culex quinquefasciatus and Aedes aegypti. DEET showed higher performance when compared to the 5x concentrated formulation and is available in the same market at a lower price than the cost of the ingredients to prepare the homemade formulation.

Conspecific and allospecific larval extracts entice mosquitoes to lay eggs and may be used in attract-and-kill control strategy.

One of the strategies of integrated vector management is to lure gravid mosquitoes for surveillance purposes or to entice them to lay eggs in water containing toxins that kill the offspring (attract-and-kill or trap-and-kill). Typically, the major challenge of this approach is the development of a lure that stimulates oviposition plus a toxin with no deterrent effect. Bacillus thuringiensis var. israelensis (Bti) satisfies the latter criterion, but lures for these autocidal gravid traps are sorely needed. We observed that gravid Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus laid significantly more eggs in cups with extracts from 4th-stage larvae (4 L) of the same or different species. No activity was found when 4 L were extracted with hexane, diethyl ether, methanol, or butanol, but activity was observed with dimethyl sulfoxide extracts. Larval extracts contained both oviposition stimulant(s)/attractant(s) and deterrent(s), which partitioned in the water and hexane phases, respectively. Lyophilized larval extracts were active after a month, but activity was reduced by keeping the sample at 4 °C. In the tested range of 0.1 to 1 larvae-equivalent per milliliter, oviposition activity increased in a dose-dependent manner. In field experiments, Ae. aegpti laid significantly more eggs in traps loaded with larval extracts plus Bti than in control traps with water plus Bti.

An unsettling explanation for the failure of skatole-baited ovitraps to capture Culex mosquitoes.

Culex mosquitoes are primarily found in temperate and tropical regions worldwide where they play a crucial role as main vectors of filarial worms and arboviruses. In Recife, a northeast city in Brazil, high densities of Culex quinquefasciatus are often found in association with human populated areas. In marked contrast to another part of the city, field tests conducted in the neighborhood of Sítio dos Pintos showed that trapping of mosquitoes in skatole-baited ovitraps did not differ significantly from captures in control (water) traps. Thus, classical and molecular taxonomic approaches were used to analyze the Culex species circulating in Sítio dos Pintos. Results obtained from both approaches agreed on the cocirculation of Culex quinquefasciatus and Culex nigripalpus in three different areas of this neighborhood. What was initially considered as an unexpected failure of this lure turned out to be a more unsettling problem, that is, the first report in Recife of Culex nigripalpus, a vector of Venezuelan equine encephalitis virus and West Nile virus. Unplanned urbanization processes close to remnants of the Atlantic forest, such as observed in Sítio dos Pintos, may have contributed to the introduction of Cx. nigripalpus in urban areas.

Development of an urban molecular xenomonitoring system for lymphatic filariasis in the Recife Metropolitan Region, Brazil.

INTRODUCTION: Molecular xenomonitoring (MX)-pathogen detection in the mosquito rather than human-is a promising tool for lymphatic filariasis (LF) surveillance. In the Recife Metropolitan Region (RMR), the last LF focus in Brazil, Culex quinquefasciatus mosquitoes have been implicated in transmitting Wuchereria bancrofti parasites. This paper presents findings on the ideal mosquito collection method, mosquito dispersion, W. bancrofti infection in mosquitoes and W. bancrofti antigen in humans to aid MX development. METHODS: Experiments occurred within two densely populated urban areas of Olinda, RMR, in July and August 2015. U.S. Centers for Disease Control and Prevention (CDC) light traps were compared to battery-powered aspirators as collection methods, and mosquito dispersion was measured by mosquito mark release recapture (MMRR). Female Cx. quinquefasciatus were tested by PCR for W. bancrofti infection, and study area residents were screened by rapid tests for W. bancrofti antigen. RESULTS: Aspirators caught 2.6 times more total Cx. quinquefasciatus, including 38 times more blood-fed and 5 times more gravid stages, than CDC light traps. They also collected 123 times more Aedes aegypti. Of the 9,644 marked mosquitoes released, only ten (0.01%) were recaptured, nine of which were < 50m (34.8m median, 85.4m maximum) from the release point. Of 9,169 unmarked mosquitoes captured in the MMR, 38.3% were unfed, 48.8% blood-fed, 5.5% semi-gravid, and 7.3% gravid. PCR on 182 pools (1,556 mosquitoes) found no evidence of W. bancrofti infection in Cx. quinquefasciatus. Rapid tests on 110 of 111 eligible residents were all negative for W. bancrofti antigen. CONCLUSIONS: Aspirators were more effective than CDC light traps at capturing Ae. aegypti and all but unfed stages of Cx. quinquefasciatus. Female Cx. quinquefasciatus traveled short (< 86m) distances in this urban area. Lack of evidence for W. bancrofti infection in mosquitoes and antigen in humans in these fine-scale studies does not indicate that LF transmission has ceased in the RMR. A MX surveillance system should consider vector-specific collection methods, mosquito dispersion, and spatial scale but also local context, environmental factors such as sanitation, and host factors such as infection prevalence and treatment history.

Field evaluation of sticky BR-OVT traps to collect culicids eggs and adult mosquitoes inside houses.

INTRODUCTION: Culex quinquefasciatus is a mosquito of importance to public health, as it represents a real and/or potential risk for the transmission of pathogens to humans, such as some arthropod-borne viruses and nematodes that cause filariasis. In Brazil, three municipalities in the Metropolitan Region of Recife (RMR) that are endemic for lymphatic filariasis conducted control actions targeting this vector. With the goal of contributing novel C. quinquefasciatus collection strategies, a sticky trap capable of collecting eggs and imprisoning mosquitoes was investigated. METHODS: To evaluate the performance of the sticky BR-OVT trap, tests were carried out in the neighborhoods of Caixa d'Água and Passarinho (Olinda-PE-Brasil) between August 2011 and June 2012. Sixty traps were installed in the indoor areas of residences in the two districts. RESULTS: During the 11-month study, 0.52 [standard deviation (SD) = 1.52] Culex egg rafts, 2.16 (SD = 4.78) C. quinquefasciatus/trap/month, and 0.55 (SD = 1.28) Aedes/trap/month were caught. Female specimens predominated the traps (59% of C. quinquefasciatus and 96% of Aedes spp.). CONCLUSIONS: The results demonstrated that the sticky BR-OVT trap is a useful tool for the collection of adult culicids of medical importance and offers an innovative way to collect C. quinquefasciatus eggs and adults in a single trap.

Field evaluation of sticky BR-OVT traps to collect culicids eggs and adult mosquitoes inside houses

Abstract INTRODUCTION: Culex quinquefasciatus is a mosquito of importance to public health, as it represents a real and/or potential risk for the transmission of pathogens to humans, such as some arthropod-borne viruses and nematodes that cause filariasis. In Brazil, three municipalities in the Metropolitan Region of Recife (RMR) that are endemic for lymphatic filariasis conducted control actions targeting this vector. With the goal of contributing novel C. quinquefasciatus collection strategies, a sticky trap capable of collecting eggs and imprisoning mosquitoes was investigated. METHODS: To evaluate the performance of the sticky BR-OVT trap, tests were carried out in the neighborhoods of Caixa d'Água and Passarinho (Olinda-PE-Brasil) between August 2011 and June 2012. Sixty traps were installed in the indoor areas of residences in the two districts. RESULTS: During the 11-month study, 0.52 [standard deviation (SD) = 1.52] Culex egg rafts, 2.16 (SD = 4.78) C. quinquefasciatus/trap/month, and 0.55 (SD = 1.28) Aedes/trap/month were caught. Female specimens predominated the traps (59% of C. quinquefasciatus and 96% of Aedes spp.). CONCLUSIONS The results demonstrated that the sticky BR-OVT trap is a useful tool for the collection of adult culicids of medical importance and offers an innovative way to collect C. quinquefasciatus eggs and adults in a single trap.

A questão regional no neoliberalismo / The regional question in neoliberalism

O texto aborda parte de pesquisa maior sobre as políticas de desenvolvimento regional nos anos recentes no Brasil, como dimensão da totalidade sistêmica que estabelece novos contornos à relação região/mundo. Esse trecho da pesquisa expõe o quadro interpretativo realizado a partir de fontes bibliográficas, documentos oficiais de governo e indicadores socioeconômicos, evidenciando que a mobilidade do capital e a externalização produtiva nesse estágio do capitalismo reconfiguram as relações de dominação nas regiões, com expansão das fronteiras dos negócios e ampliação da mercantilização da reprodução social, o que aprofunda a degradação do trabalho e a precariedade das condições urbanas.

The text covers part of most research on regional development policies in recent years in Brazil, as a dimension of systemic totality establishing new contours local/global relationship. This research section exposes the interpretive framework made from literature sources, official documents of government and socio-economic indicators, showing that the mobility of capital and the productive externalization of this stage of capitalism reconfigure the relations of domination in regions with expanding borders of the business and commodification expansion of social reproduction, which deepens the degradation of work and the precariousness of urban conditions.

Insecticide susceptibility of Aedes albopictus and Ae. aegypti from Brazil and the Swiss-Italian border region.

BACKGROUND: Aedes aegypti and Ae. albopictus are two highly invasive mosquito species, both vectors of several viruses, including dengue, chikungunya and Zika. While Ae. aegypti is the primary vector in the tropics and sub-tropics, Ae. albopictus is increasingly under the public health watch as it has been implicated in arbovirus-transmission in more temperate regions, including continental Europe. Vector control using insecticides is the pillar of most control programmes; hence development of insecticide resistance is of great concern. As part of a Brazilian-Swiss Joint Research Programme we set out to assess whether there are any signs of existing or incipient insecticide resistance primarily against the larvicide Bacillus thuringiensis svar. israelensis (Bti), but also against currently applied and potentially alternative insecticides in our areas, Recife (Brazil) and the Swiss-Italian border region. METHODS: Following World Health Organization guidelines, dose-response curves for a range of insecticides were established for both colonized and field caught Ae. aegypti and Ae. albopictus. The larvicides included Bti, two of its toxins, Cry11Aa and Cry4Ba, Lysinibacillus sphaericus, Vectomax CG®, a formulated combination of Bti and L. sphaericus, and diflubenzuron. In addition to the larvicides, the Swiss-Italian Ae. albopictus populations were also tested against five adulticides (bendiocarb, dichlorodiphenyltrichloroethane, malathion, permethrin and λ-cyhalothrin). RESULTS: Showing a similar dose-response, all mosquito populations were fully susceptible to the larvicides tested and, in particular, to Bti which is currently used both in Brazil and Switzerland. In addition, there were no signs of incipient resistance against Bti as larvae were equally susceptible to the individual toxins, Cry11Aa and Cry4Ba. The field-caught Swiss-Italian populations were susceptible to the adulticides tested but DDT mortality rates showed signs of reduced susceptibility. CONCLUSIONS: The insecticides currently used for mosquito control in Switzerland and Brazil are still effective against the target populations. The present study provides an important reference as relatively few insecticide susceptibility surveys have been carried out with Ae. albopictus.

Zika virus replication in the mosquito Culex quinquefasciatus in Brazil.

Zika virus (ZIKV) is a flavivirus that has recently been associated with an increased incidence of neonatal microcephaly and other neurological disorders. The virus is primarily transmitted by mosquito bite, although other routes of infection have been implicated in some cases. The Aedes aegypti mosquito is considered to be the main vector to humans worldwide; however, there is evidence that other mosquito species, including Culex quinquefasciatus, transmit the virus. To test the potential of Cx. quinquefasciatus to transmit ZIKV, we experimentally compared the vector competence of laboratory-reared Ae. aegypti and Cx. quinquefasciatus. Interestingly, we were able to detect the presence of ZIKV in the midgut, salivary glands and saliva of artificially fed Cx. quinquefasciatus. In addition, we collected ZIKV-infected Cx. quinquefasciatus from urban areas with high microcephaly incidence in Recife, Brazil. Corroborating our experimental data from artificially fed mosquitoes, ZIKV was isolated from field-caught Cx. quinquefasciatus, and its genome was partially sequenced. Collectively, these findings indicate that there may be a wider range of ZIKV vectors than anticipated.

Does Zika virus infection affect mosquito response to repellents?

The World Health Organization (WHO) recommends that people travelling to or living in areas with Zika virus (ZIKV) outbreaks or epidemics adopt prophylactic measures to reduce or eliminate mosquito bites, including the use of insect repellents. It is, however, unknown whether repellents are effective against ZIKV-infected mosquitoes, in part because of the ethical concerns related to exposing a human subject's arm to infected mosquitoes in the standard arm-in-cage assay. We used a previously developed, human subject-free behavioural assay, which mimics a human subject to evaluate the top two recommended insect repellents. Our measurements showed that DEET provided significantly higher protection than picaridin provided against noninfected, host-seeking females of the southern house mosquito, Culex quinquefasciatus, and the yellow fever mosquito, Aedes aegypti. When tested at lower doses, we observed a significant reduction in DEET-elicited protection against ZIKV-infected yellow fever mosquitoes from old and recent laboratory colonies. The reduction in protection is more likely associated with aging than the virus infection and could be compensated by applying a 5x higher dose of DEET. A substantial protection against ZIKV-infected and old noninfected mosquitoes was achieved with 5% DEET, which corresponds approximately to a 30% dose in the conventional arm-in-cage assays.

Tecnologias integradas para controle biológico, mecânico e genético de Aedes aegypti / Integrated technologies for biological, mechanical and genetic control of Aedes aegypti

Duas tecnologias alternativas para o controle de Aedes aegypti foram avaliadas: a aplicação espacial de larvicida biológico-Bti em potenciais criadouros peridomiciliares, e a liberação de machos estéreis para inviabilização reprodutiva das fêmeas do mosquito. As ações foram realizadas pelos Agentes dos Serviços de Saúde em 15 vilas da Ilha de Fernando de Noronha, e em uma área (900 imóveis) no bairro da Várzea/Recife/Pernambuco. A efetividade dos métodos foi avaliada por indicadores entomológicos,estimados pela presença, quantidade e viabilidade de ovos do mosquito, coletados em armadilhas, e por marcadores genéticos. A aplicação de Bti, com atomizador costal, ocorreu a cada 30 dias em ambas as áreas. Uma redução importante e sustentável da população de A. aegypti,por este método, foi alcançada em 2015/2016 na Várzea e, em 2016, na Ilha, onde a remoção de 18 toneladas de resíduos sólidos em 2015 contribuiu possivelmente para os resultados. Machos esterilizados com radiação gama foram produzidos em massa no laboratório e liberados em uma das vilas da Ilha. A análise espaço-temporal dos indicadores, de dez/2015a ago/2016, revelou redução expressiva da densidade populacional do mosquito e da diversidade genética da população local. Ambas as abordagens parecem ter reduzido o contato homem-vetor e os riscos de transmissão de arboviroses na Ilha, apesar da elevada competência vetorial da população local do mosquito para os vírus Zika e Dengue. Os métodos testados se mostraram eficientes e passíveis de serem integradas às ações do SUS voltadas ao controle de A. aegypti.

Two alternative technologies were evaluated for Aedes aegypti control:the spraying of a biological larvicide (Bti) in potential peridomiciliarybreeding sites and the release of sterile males to promote reproductionblockage in wild females. Actions were carried out by Agents of theHealth Services, in 15 villages of the Fernando de Noronha Island and in 900 properties from the district of Várzea, Recife-PE. The effectiveness of both methods was evaluated by entomological indicators, estimatedby the presence, quantity and viability of eggs from the mosquito collectedin traps and through genetic markers. Bti was delivered by backpacksprayer every 30 days in both areas. A significant and sustainablereduction of the A. aegypti population as a result of this technique wasachieved in 2015/2016 in Várzea and in 2016 in the Island, where it wasstrengthened by the removal of 18 tons of solid waste in 2015. Malessterilized with gamma radiation were mass-produced in the laboratoryand released in one village of the Island. The spatiotemporal analysis ofthe indicators, from Dec/2015 to Aug/2016, revealed a significant reductionin mosquito density, which impacted on the genetic diversity of thelocal population. Both approaches seem to have reduced human-vectorcontact and the risk of arbovirus transmission in the Island, althoughlocal mosquito population presented high vector competence to Zikaand Dengue virus. These methods were efficient and could be integratedinto SUS actions directed to A. aegypti control.

Arranjo produtivo local, política do espaço e flexibilização do trabalho / Local production arrangement, space policy and flexiwork

Resumo: Este artigo analisa a política nacional para Arranjo Produtivo Local (APL) nos governos do Partido dos Trabalhadores (PT). Baseia-se em investigação qualitativa de documentos governamentais e títulos bibliográficos, demonstrando que a política pública visa mobilizar condicionalidades para alargar as fronteiras do capital em sua crise estrutural, ao mesmo tempo em que o APL se estrutura como um aparato ideológico estratégico para coalizão de classes nas regiões, encobrindo a desigualdade com o fetiche do desenvolvimento local para todos.

Abstract: In this article it is analyzed the national policy for Local Production Arrangement (Arranjo Produtivo Local - APL) in the Workers' Party's (Partido dos Trabalhadores - PT) governments. The analysis is based on the qualitative investigation of governmental documents and bibliography data, and it shows that the public policy aims at mobilizing conditions to extend the capital boundaries, in its structural crisis, at the same time that APL structures itself as a strategical ideological apparatus directing to the coalition of regional social classes, thus covering-up the inequality with the charming idea of local development for everybody.

Milk traits of lactating cows submitted to feed restriction.

Data from five experiments with dairy cows where feed was restricted to 0, 40, and 50% of the ad libitum amount, with 259 observations, were subjected to multivariate analyses to determine the effects of severity and duration of feed restriction on production, physical-chemical characteristics, ethanol stability, and somatic cell score of milk. A negative relationship was seen between the severity and duration of feed restriction with milk production, lactose content, titratable acidity, and milk stability to the ethanol test. The milk stability to the ethanol test, protein content, milk yield, and somatic cells score were the most important attributes retained by the discriminant analysis. Milk stability to the ethanol test, live weight, days in restriction, and pH were the most important characteristics explaining the variance within the different levels of feed restriction. Milk production and ethanol stability were significantly lower in both levels of feed restriction compared with the group fed ad libitum. When feed restriction was followed by refeeding, the difference observed in ethanol stability was the first discriminant variable, followed by the difference in unstable milk frequency and titratable acidity. Increments in the severity and duration of feed restriction negatively affect milk production and milk ethanol stability.