Phlebotominae (Diptera, Psychodidae) na Província Espeleológica do Vale do Ribeira - 1. Parque Estadual Intervales (PEI), estado de São Paulo, Brasil

The identification of the sandfly fauna and investigation of some ecological aspects of its populations in areas frequented by tourists of the PEI, an Atlantic forest reserve with many caves, were the objective of this study. Captures were undertaken monthly from January 2001 to December 2002, with automatic light traps installed in 13 ecotopes, including caves, forests, domiciliary and peridomiciliary environments, and by aspiration in armadillo burrows. Additionally, although not at regular intervals, Shannon traps were installed in forests and anthropic environments, aspirations were made on cave walls, among roots and fallen leaves, and some insects were captured while biting researchers. A total of 891 sandflies belonging to 21 species were captured. Six hundred specimens representing 19 species were captured with light traps, 215 in anthropic (2.24 insects/trap) and 385 in extra-domiciliary (1.46 insects/trap) environments. Brumptomyia troglodytes was the most abundant species (the Standardised Index of Species Abundance = 0.705). Pintomyia monticola predominated in the Shannon traps and showed anthropophilic and diurnal activity. Psathyromyia pascalei predominated in the aspirations; the largest number being in armadillo burrows. Eleven species were captured in caves; although some might be troglophiles, the majority used these ecotopes as resting places. Nyssomyia intermedia, Nyssomyia neivai and Migonemyia migonei, implicated in the transmission of cutaneous leishmaniasis in the Southeastern Brazilian region, were all found, though in such low densities as to suggest minimal risk of the disease in the PEI.A identificação da fauna flebotomínea e de alguns aspectos ecológicos de suas populações em áreas freqüentadas por turistas no PEI, situado em reserva de mata Atlântica, constituem-se nos objetivos deste estudo. As capturas foram mensais de janeiro/2001 a dezembro/2002, com armadilhas automáticas luminosas em 13 ecótopos, incluindo cavernas, matas e peridomicílio e aspiração em tocas de tatus. Sem intervalos regulares, foram realizadas capturas com armadilhas de Shannon na mata e ambientes antrópicos, aspiração em paredes de cavernas e entre raízes e folhedo e em membros da equipe quando picados por flebotomíneos. No total foram capturados 891 flebotomíneos pertencentes a 21 espécies. Com as armadilhas automáticas luminosas capturou-se 19 espécies e 600 espécimes, 215 em ambiente antrópico (2,24 insetos/armadilha) e 385 em ambiente natural (1,46 insetos/armadilha). Brumptomyia troglodytes foi a mais abundante, com o índice de abundância das espécies padronizado = 0,705. Pintomyia monticola predominou nas armadilhas de Shannon, mostrando-se antropofílica e com atividade hematofágica diurna e noturna. Psathyromyia pascalei predominou nas aspirações, com a maioria dos espécimes aspirados de tocas de tatu. Das 11 espécies capturadas em cavernas, embora algumas possam ser consideradas troglófilas, a maioria usa este ecótopo como local de abrigo. Nyssomyia intermedia, Nyssomyia neivai e Migonemyia migonei, implicados na transmissão da leishmaniose tegumentar na Região Sudeste do Brasil foram capturados, todavia em tão baixa densidade que sugere risco mínimo da doença no PEI.FAPESPIBAMA-S

Ecology of Haemagogus and Sabethes (Diptera: Culicidae) Mosquitoes in Epizootic Yellow Fever Regions of Rio Grande do Sul State, Brazil

Objetivou-se com este estudo investigar característica ecológica e o hábito alimentar de Haemagogus e Sabethes em área epizoótica da febre amarela silvestre em duas localidades do Rio Grande do Sul, capturados com aspirador, armadilha de Shannon e puçá. Para identificação do sangue ingerido pelas fêmeas foi utilizada a técnica imunoenzimática ELISA de captura. Obteve-se maior atividade de Hg. leucocelaenus na primavera e outono, entre 12 e 17 horas, enquanto Sabethes albiprivus e Sa. quasycianus durante todo o ano. Para fêmeas de Hg. leucocelaenus em Santo Antônio das Missões, houve predomínio de sangue humano e para as demais espécies destaca-se a atratividade para bovinos nos dois municípios. A capacidade vetorial do Hg. leucocelaenus fundamenta vigilância entomológica em seu papel de estratificar áreas problemas ou a sustentabilidade do programa de imunização da populaçãoThe study was conducted in epizootic areas of sylvatic yellow fever in two municipalities in Rio Grande do Sul State, with the objective to investigate ecological characteristics of Haemagogus and Sabethes mosquitoes. Mosquitoes were collected using Shannon traps and insect nets. The host-feeding pattern was tested by an enzyme-linked immunosorbent assay. The highest activity levels were observed for Hg. leucocelaenus during spring and autumn, between 12 and 17 hours, while Sabethes albiprivus and Sa. quasycianus were common throughout the year. The blood of females of Hg. leucocelaenus at one municipality, Santo Antonio das Missoes, was of human origin. Considering the other species, in both municipalities of the study, bovine blood was predominant. The vectorial capacity of Hg. leucocelaenus justifies the entomological surveillance in order to stratify problematic areas or the sustainability of an immunization programSecretaria de Vigilância em Saúde do Ministério da Saúde, OPAS/OM

Standardizing a enzime-linked immunossorbent assay (ELISA) to be employed in studies related to food habits of Diptera

A identificação de sangue ingerido pelos insetos constitui-se em importante parâmetro para elucidar aspectos ligados à transmissão de zoonoses, dentre elas, as leishmanioses. Dentre alguns métodos empregados para esclarecer a atração de vetores por animais que possam atuar como reservatórios dessas parasitoses, destacam-se os métodos sorológicos. O presente estudo teve como objetivo, padronizar a técnica imunoenzimática (ELISA) de captura, a ser utilizada em pesquisas relacionadas à atração de vetores das leishmanioses. Para a padronização da técnica, foram utilizadas fêmeas de flebotomíneos ingurgitadas da espécie Lutzomyia longipalpis, criadas em laboratório em temperatura ambiente de 25 ± 2°C, alimentadas experimentalmente em rato e com a utilização de reagentes disponíveis no comércio. Em vista da alta sensibilidade, favorecida pelo sistema avidina-biotina, quantificou-se sangue ingerido para amostras com diferentes períodos de digestão, utilizando-se a menor concentração de anti-soro. A técnica pôde prover a realização de pelo menos noventa testes em duplicata para a determinação de sangue recém-ingerido. Para todas as amostras com períodos de 12 e 24 horas pós-ingestão, foi constatada a presença de sangue, observando-se diferença significativa entre os respectivos títulos, com a possibilidade de detecção de sangue até 48 horas e observada a ausência total de reação após 72 horas da ingestão de sangue.Blood meals taken by insects constitute an important parameter for the elucidation of aspects of the transmission of the zoonoses, and among them, the Leishmaniases. Among the methods used in the investigation of the attraction of those animals, wich may be hosts of these parasitoses, exercised over vectors, are the immunological assays. This study was undertaken for the purpose of standardizing a sandwich enzime-linked immunossorbent assay (ELISA) to be employed in research projects related to leishmaniases vectors attraction to animals. Laboratory-bred sandflies, Lutzomyia longipalpis, maintained at 25± 2°C, and comercial reagents, were employed. fed on rats and In the light of the high sensibility that the biotin-avidin method permits, samples with different time periods of digestion were quantified, by means of the smallest concentration of antisera. The technique provided at least ninety repeat tests to determine recent blood meals taken by these insects. Blood meals were detectable up to 12 and 24h after feeding, and a significant difference between these titles was observed, with the possibility of detecting blood meals up to 48h after ingestion and the total absence of reaction 72 h thereafter

Brumptomyia carvalheiroi Shimabukuro & Galati, sp.nov.

Brumptomyia carvalheiroi Shimabukuro & Galati, sp.nov. (Figs 1-17, 19) Holotype (male): total body length: 3388 (3708 ± 231; n = 10). Predominantly brown, with brown pronotum, paratergite and mesonotum, and light brown thoracic pleura and coxae, but without clear contrast. Head (Fig. 1): length 435 (425 ± 20; n = 10); width 400 (409 ± 15; n = 10). Eyes: length 278 (273 ± 20; n = 10), width 148 (148 ± 15; n = 10) (frontal view). Interocular distance 119 (120 ± 7; n = 10). Interocular suture united to the antennal suture. Clypeus length: 119 (119 ± 7; n = 10). Antennomere lengths: AIII (Fig. 3) 435 (424 ± 19; n = 10), AIV (Fig. 4) 209 (206 ± 10; n = 10), AV (Fig. 4) 202 (204 ± 11; n = 10), AXV (Fig. 6) 101 (95 ± 6; n = 10) and AXVI (Fig. 6) 73 (73 ± 5; n = 10). Antennal formula AIII – AXV 2, AXVI 0; ascoids with posterior spur, anterior prolongation long reaching the distal end of the flagellomere; papilla pre-apical present on AIII - AV (Figs. 3 and 4) and AXIII (Fig. 5). Length of the palpomeres: I 55 (54 ± 3; n = 10), II 132 (128 ± 10; n = 10), III 172 (167 ± 9; n = 10), IV 154 (148 ± 11; n = 10), V 391 (368 ± 20; n = 10). Palpal formula: 1.2.4.3. 5. Newstead’s spines on the basal half of palpomere III and absent from palpomere II. Labroepipharynx 244 (240 ± 11; n = 10) long. Labial sutures united. Cibarium reduced without teeth (Fig. 11). Cervix: ventro-cervical sensilla present. Thorax: mesonotum length 626 (651 ± 39; n = 10). Pleura with 7 proepimeral setae (3–7; n = 10) and 14 upper anepisternal setae (10–18; n = 10) and without metaepisternal setae. Setae present on the anterior katepisternum margin. Incomplete suture between katepimeron and metepisternum present. Wing (Fig. 13): length 2705 (2765 ± 215; n = 10), width 895 (910 ± 81; n = 10). Length of vein sections: alpha 696 (705 ± 84; n = 10), beta 348 (490 ± 300; n = 10), gamma 661 (591 ± 188; n = 10), delta 104 (108 ± 23; n = 10), pi 43 (26 ± 11; n = 10), R 5 1747 (1710 ± 72; n = 10). Length of femora, tibiae, basitarsi and tarsi II+III+IV+V: foreleg 937 (999 ± 51; n = 10), 1406 (1376 ± 113; n = 10), 809 (786 ± 70; n = 10), 873 (967 ± 58; n = 10); midleg 916 (993 ± 50; n = 10), 1278 (1553 ± 128; n = 10), 852 (882 ± 47; n = 10), 916 (980 ± 41; n = 10); hindleg 1083 (1129 ± 49; n = 10), 1810 (1883 ± 82; n = 10), 980 (1020 ± 89; n = 10), 1065 (1069 ± 48; n = 10). Abdomen 1917 (2239 ± 45; n = 10) long. Tergal papilla absent on all tergites. Terminalia (Fig. 17): gonostyle 357 (339 ± 15; n = 10) long, with 5 spines; the spines having the following disposition: two apical, both the external located on a single tubercle, the internal at the same level than the external ones, on the middle of the gonostyle. Gonocoxite (679 long x 52 wide) (706 ± 21 x 70 ± 12; n = 10), with a basal tuft of thin long setae located on a tubercle, apex bearing four long spines. Paramere long and simple, dorsal margin length 242 (224 ± 33; n = 10) and the ventral margin length 345 (363 ± 20; n = 10) with thin setae along its lateral length and some stronger setae on its apex (Fig. 19). Triangular aedeagus, dorsal margin length 101 (108 ± 10; n = 10) and ventral margin length 103 (96 ± 15; n = 10). Lateral lobe length 731 (717 ± 31; n = 10), width 21 (26 ± 2; n = 10). Genital pump: 130 (154 ± 13; n = 10) long; piston length 103 (121 ± 10; n = 10) and chamber length 55 (61 ± 5; n = 10). Genital filaments length 3001 (2836 ± 476; n = 10) or more than 20 times the length of genital pump (17 ± 4; n = 10). Slender genital ducts. Cercus 244 long (252 ± 14; n = 10). Alotype (female): total body length 3634 (3586 ± 263; n = 10). General coloration as for the male. Head (Fig. 2): length 470 (449 ± 20; n = 10), width 435 (429 ± 14; n = 10). Eyes: length 270 (275 ± 14; n = 10), width 157 (156 ± 10; n = 10) (frontal view). Interocular suture united to the antennal suture. Interocular distance 139 (129 ± 7; n = 10). Clypeus 139 (135 ± 6; n = 10) long. Flagellomeres: AIII (Fig. 7) 339 (337 ± 16; n = 10), AIV (Fig. 8) 163 (158 ± 10; n = 10), AV 169 (Fig. 8) (162 ± 12; n = 10), AXV (Fig. 10) 90 (91 ± 5; n = 10), and AXVI (Fig. 10) 70 (69 ± 8; n = 10). Ascoids with posterior spur and the anterior prolongation, long, reaching or almost reaching the next flagellomere (Fig. 7). Papilla present on AV (Fig. 8) and AXIII (Fig. 9). Palpomere lengths: I 59 (57 ± 4; n = 10), II 138 (137 ± 8; n = 10), III 165 (166 ± 10; n = 10), IV 152 (148 ± 7; n = 10), V 383 (367 ± 17; n = 17). Palpal formula: 1.2.4.3. 5. Newstead’s spines (about 10) distributed throughout the middle of palpomere III. Labroepipharynx 296 (272 ± 32; n = 10) long. Cibarium (Fig. 12) with unusual numerous anterior teeth horizontally and in lateral position, forming some rows, the posterior (horizontal) teeth; pigment patch poorly developed and posterior bulge well developed; arch incomplete. Pharynx unarmed. Labial sutures united. Maxilla: lacinia with 7 external teeth disposed in a longitudinal row and about 18 internal teeth. Hypopharynx with apicolateral teeth well delineated. Cervix: ventrocervical sensilla present. Thorax: mesonotum 757 (729 ± 44; n = 10) long. Pleurae with 7 proepimeral setae (4–7; n = 10), 15 upper anepisternal setae (12–21; n = 10) and without metaepisternal setae. Setae present on the anterior katepisternum margin. Incomplete suture between katepimeron and metaepisternum present. Wing (Fig. 14): length 2939 (2806 ± 112; n = 10) and width 1001 (920 ± 54; n = 10). Length of vein sections: alpha 726 (692 ± 32; n = 10), beta 366 (346 ± 37; n = 10), gamma 504 (602 ± 322; n = 10), delta 174 (123 ± 32; n = 10), pi 35 (46 ± 27; n = 10), R 5 1874 (1797 ± 107; n = 10). Length of femora, tibiae, basitarsi and tarsi II+III+IV+V: 1065 (1024 ± 61; n = 10), 1257 (11228 ± 69; n = 10), 724 (726 ± 27; n = 10), 958 (946 ± 49; n = 10); midleg 1001 (1033 ± 64; n = 10), 11459 (1466 ± 78; n = 10), 809 (824 ± 61; n = 10), 1044 (987 ± 49; n = 10; hindleg 11171 (1161 ± 64; n = 10), 1810 (1672 ± 338; n = 10), 958 (939 ± 111; n = 10), 1108 (1054 ± 82; n = 10). Abdomen length 2407 (2408 ± 245; n = 10). Tergite VIII with 2 setae. Spermathecae body (Fig. 15) with 25-40 + annuli (66 long x 18 wide) (69 ± 9 x 16 ± 2; n = 10), the apical ring being wider than the basal one; smooth individual ducts, common duct apical width (Fig. 16) 28 ± 6; n = 3, basal width: 43 ± 0; n = 3 and length: 497 ± 32; n = 3. Cercus 141 long (143 ± 3; n = 10) and 68 wide (55 ± 9; n = 10). Type data. Holotype male. BRAZIL, São Paulo, Iporanga, Parque Estadual Turístico do Alto Ribeira (PETAR), Igreja, A. Paiva, 09.x. 2002: mALT, 16 h- 7 h. Alotype female. BRAZIL, São Paulo, Ribeirão Grande, Parque Intervales, Colorida Cave near the entrance, 13-14.xii. 2002, ALTm, 18 h- 7 h (FSP/ USP). Paratypes, 14 males and 14 females (FSP/ USP): 1 male: Ribeirão Grande, Parque Intervales, peridomicile, 26-27.x. 2001, mALT; 1 male: Ribeirão Grande, Parque Intervales, armadillo burrow 1, 21–22.ix. 2001, morning aspiration; 1 male and 1 female: Ribeirão Grande (Parque Intervales), armadillo burrow, 26.v. 2001, morning aspiration; 2 males and 3 females: Ribeirão Grande, Parque Intervales, Colorida Cave near the entrance, 14.xii. 2002, mALT; 1 male: Ribeirão Grande, Parque Intervales, inside Colorida Cave, 13–14.xii. 2002, Shannon Trap; 1 male: Iporanga, Parque Estadual Turístico do Alto Ribeira (PETAR), top of Morro Preto Cave, 16- 17.xii. 2001, mALT; 2 males: Ribeirão Grande, Parque Intervales, Colorida Cave near the entrance, 13.xii. 02002, aspiration; 2 males and 2 females: Ribeirão Grande, Parque Intervales, Colorida Cave near the entrance, 13-14.xii. 2002, ALTm, 18 h- 7 h; 1 male and 1 female: Ribeirão Grande, Parque Intervales, Colorida Cave ground, 28-29.xi. 2002, Shannon Trap; 2 males: Iporanga, Parque Estadual Turístico do Alto Ribeira, (PETAR), Igreja, armadillo burrow, 10.x. 2002, aspiration, A. Paiva, 13 h; 2 females: Ribeirão Grande, Parque Intervales, armadillo burrow 1, 22.ix. 2001, aspiration; 1 female: Ribeirão Grande, Parque Intervales, Colorida Cave near the entrance, 28-29.xi. 2002, aspiration; 1 female: Ribeirão Grande, Parque Intervales, Colorida Cave near the entrance, 14.xii. 2002, aspiration; 1 female: Ribeirão Grande, Parque Intervales, Colorida Cave near the entrance 2, 18–19.ii. 2001, mALT, 19 h– 7 h; 2 females: Ribeirão Grande, Parque Intervales, peridomicile, 18–19.x. 2002, Shannon Trap. The type material was captured by E. A. B. Galati, A. M. Marassá, R. M. Gonçalves- Andrade and A. Galati. Etymology. We are pleased to name this species after Dr. José da Rocha Carvalheiro (Fiocruz) for his contributions to Public Health and Epidemiology in Brazil. Taxonomic Discussion. Among the Brumptomyia species in which the males possess a style, with the internal spine implanted at the same level as the external ones and the gonocoxite possessing a basal tuft of setae implanted in a small tubercle, such as are found in B. carvalheiroi sp. nov., are B. mangabeirai (Barretto & Coutinho, 1941), B. pentacantha (Barretto, 1947), B. galindoi (Fairchild & Hertig, 1947), B. mesai Sherlock, 1962 (revalidated: Ibanez-Bernal, 1999). Compared to these three species, the new taxon B. carvalheiroi is most similar to B. mangabeirai (Fig. 18). However, B. carvalheiroi and B. mangabeirai may be distinguished from one another by the shape of the paramere (Fig. 20), which is wider at the base in B. carvalheiroi (at least two times the apical width) (2 ± 0.35; n = 10) compared to B. mangabeirai (less than two times the apical width) (1.7 ± 0.36; n = 10) (Figs. 19, 20). The mean values for the head, palpi, AXV, AXVI, mesonotum and the wing indexes beta and gama and gonocoxite length are higher in B. carvalheiroi sp. n., whilst the head width, lengths of AIII, AIV, AV, the wing indexes alfa and delta, gonocoxite width, lengths of gonostyle, paramere and lateral lobes have higher mean values in B. mangabeirai (Barretto & Coutinho, 1941). B. carvalheiroi can be distinguished from B. pentacantha because in the latter species the setae of the basal tuft are clearly shorter, the paramere has a different shape and there are two papillae on the AIII. B. carvalheiroi can be distinguished from B. galindoi because the lower setae of the basal tuft of the gonocoxite are semifociaceous in the latter species and the ejaculatory ducts are much shorter. B. carvalheiroi can be distinguished from B. mesai because the setae of the basal tuft of the gonocoxite are semifoliaceous and the ejaculatory ducts are much shorter in the latter species. Among the species of Brumptomyi a described by their females, the new species B. carvalheiroi may be easily distinguished by (i) the strikingly small and numerous anterior and lateral teeth in the cibarium and (ii) spermathecae with very long individual sperm ducts. Thus, B. carvalheiroi can be distinguished from females of B. hamata by the ratio of the lengths of the body of the spermathecae to the individual ducts of the spermathecae, which is less than 4 in the latter and more than ten times in the former species. B. carvalheiroi can also be distinguished from B. pentacantha and B. avellari because the latter two species possess two papillae on the AIII, while the former species has only one pre-apical papilla in AIII. B. carvalheiroi can be distinguished from B. leopoldoi, B. mesai and B. brumpti by the number of rings on the spermathecae body, ca. 10 in B. leopoldoi, ca. 15 in B. mesai, less than 20 in B. brumpti and more than 25 (25–40 +) in B. carvalheiroi. Finally, B. carvalheiroi can also be distinguished from B. nitzulescui, which also possesses a similar number of spermatheca rings, because the individual sperm ducts of the latter species possess several turns, with short sinuosities along their course (“sianinha” aspect).Published as part of Shimabukuro, Paloma Helena Fernandes, Marassá, Ana Maria & Apare-, Eunice, 2007, Brumptomyia carvalheiroi sp. nov. (Diptera: Psychodidae: Phlebotominae) from Atlantic forest domain, São Paulo State, Brazil, pp. 47-54 in Zootaxa 1637 on pages 48-54, DOI: 10.5281/zenodo.17957

The subgenus Migonemyia Galati 1995 (Diptera, Psychodidae, Phlebotominae), with description of a new species Migonemyia vaniae: a review

The capture of a new species of the subgenus Migonemyia Galati, 1995 (Diptera, Psychodidae, Phlebotominae), Migonemyia vaniae sp. nov. in the Ribeira Valley, state of São Paulo, Brazil, together with the other two species: Mg. migonei (França, 1920) and Mg. rabelloi (Galati &amp; Gomes, 1992) lead us to review this subgenus. The new species was described and illustrated. The genitalia of the two other species were also illustrated and some genital characteristics (number of setae on the gonocoxite tuft, ejaculatory ducts and pump and ducts/pump ratio; and number of setae on the tergite VIII of the females) considered important to differentiate the three species, including five populations of Mg. migonei (from Northeastern, Southeastern, and Southern Brazilian regions and of Peru) were submitted to variance analyses. The Mg. migonei population of Northeastern Brazilian region showed distinct smaller values (P < 0.05) than the other Brazilian populations studied as regarding these characteristics. The capture of both sexes of these three species in sympatry confirms the association between the sexes of Mg. rabelloi, recognised as doubtful when this species was originally described. Identification keys for male and female of the three species are presented

Padronização da técnica imunoenzimática do ELISA de captura, no sistema avidina-biotina para a identificação de sangue ingerido por Lutzomyia (Lutzomyia) longipalpis (Lutz & Neiva, 1912) Enzyme-linked Immunosorbent Assay biotin/avidin method standardization, for identification of Lutzomyia (Lutzomyia) longipalpis bloodmeals (Lutz & Neiva, 1912)

A identificação de sangue ingerido pelos insetos é um importante parâmetro para elucidar aspectos ligados à transmissão de zoonoses, dentre elas, as leishmanioses. Dos métodos empregados para esclarecer a atração de vetores por animais que possam atuar como reservatórios dessas parasitoses, destacam-se os imunológicos. O estudo teve como objetivo, padronizar a técnica imunoenzimática de captura e titular amostras de sangue ingerido em fêmeas de flebotomíneos ingurgitadas de Lutzomyia longipalpis criadas em laboratório e alimentadas experimentalmente em rato. Em vista da alta sensibilidade, favorecida pelo sistema avidina-biotina, foi possível a realização de pelo menos noventa testes, de cada uma das amostras em duplicata, e constatar a presença de sangue para todas as amostras com períodos de 12 e 24 horas pós-ingestão, observando-se diferença significativa entre os respectivos títulos.Bloodmeals taken by insects constitute an important parameter for clarifying aspects of the transmission of zoonoses, including leishmaniases. Immunological assays can be used to investigate the attraction of vectors to animals, which may be hosts of these parasitoses. The objective of this study was to standardize a sandwich enzyme-linked immunosorbent assay and titer samples with different time periods of digestion, in laboratory-bred Lutzomyia longipalpis fed on rats. In the light of the high sensitivity that the biotin-avidin method permits, the technique provided at least ninety repeat tests for each sample and identified recent bloodmeals taken by these insects. Bloodmeals were detectable up to 12 and 24h after blood ingestion, and a significant difference between these titers was observed