Experimental study of the infecting ability of the flea Coptopsylla lamellifer rostrata in the Kyzylkum natural focus of plague

Experimental infecting of the fleas Coptopsylla lamellifer rostrata by plague was carried out. The fleas were infected by feeding through biomembrane (skin of a white mouse) with a mixture of defibrinated blood of guinea-pig and plague microbes. Under concentration 2-3 milliard of the microbes in 1 millilitre, from 60 to 100% of the fleas were infected. Forming of the block of proventriculus was observed in 9-13 day. Mean percent of blocking for all experiments was less than 1%. Maximal rate of blocking (2.8%) was observed in the experiment under everyday 3-hour feeding. After planting of the fleas with blocks on great gerbil (2 fleas on one gerbil) 20% of the gerbils were infected. The conclusion had been made that C. lamellifer rostrata has only insignificant effectiveness as a vector of plague agent in Kyzylkum. Probably it is caused by some specific features of this subspecies.     

Characteristics of digestion of blood of different animals by the flea Xenopsylla cheopis]

Under experimental conditions fleas X. cheopis engorged and assimilated blood of 4 species of rodents, man, pigeon and two species of reptiles. Histological investigations have shown that the structure of food clot, destruction rate of blood cells and general duration of digestion are changed considerably depending on the host's blood. In females the digestion of white mice blood to haematin at 23-24 degrees lasted not over 12 hours, the digestion of blood of hamsters and man - 12 to 20 hours, of guinea pigs and reptiles - 18 to 25 hours and of white rats and pigeons - from 20 to 30 hours. In males the digestion carries out somewhat slower. The above differences in blood digestion can apparently create unequal conditions for pathogenic microbes in the intestine of fleas that affects the preservation and reproduction of the agent in the organisms of these carriers.     

Block formation capacity in fleas infected with a plague agent after their feeding with blood substitutes

By feeding plague infected Xenopsylla cheopis fleas with blood substitutes free of blood formed elements blocked fleas were obtained. These fleas are able to transmit plague agent to white mice and cause their mortality.     

Effect of the digestive process on the survival of the plague agent in Xenopsylla gerbilli Minax fleas

Hystological investigations of experimentally infected X. gerbilli minax have shown that at the early stages of the digestion, while in the intestine compact clots of non-digested blood, are preserved, proceeds the reproduction of the plague agent. With the decay of the alimentary clot it changes into the dying off of the microbe population and after the ceasing of digestion the accumulation of bacteria renews again. Simultaneously with the changes in the number of microbes their morphology varies. At the initial stage of blood digestion bacilli typical for the agent are dominant. With the decay of the alimentary clot they are replaced by ovoid and spheroid forms. After the ceasing of the digestion cycle the microbe mass is represented by small coccobacteria. During the decrease in the plague agent abundance bacteria are absent mostly often from the midgut and posterior parts of the alimentary canal. Most favourable conditions for their preservation exist in this period in the proventriculus and in adjoining to it oesophagus part.     

Similarities and seasonal variations in bacterial communities from the blood of rodents and from their flea vectors

Vector-borne microbes are subject to the ecological constraints of two distinct microenvironments: that in the arthropod vector and that in the blood of its vertebrate host. Because the structure of bacterial communities in these two microenvironments may substantially affect the abundance of vector-borne microbes, it is important to understand the relationship between bacterial communities in both microenvironments and the determinants that shape them. We used pyrosequencing analyses to compare the structure of bacterial communities in Synosternus cleopatrae fleas and in the blood of their Gerbillus andersoni hosts. We also monitored the interindividual and seasonal variability in these bacterial communities by sampling the same individual wild rodents during the spring and again during the summer. We show that the bacterial communities in each sample type (blood, female flea or male flea) had a similar phylotype composition among host individuals, but exhibited seasonal variability that was not directly associated with host characteristics. The structure of bacterial communities in male fleas and in the blood of their rodent hosts was remarkably similar and was dominated by flea-borne Bartonella and Mycoplasma phylotypes. A lower abundance of flea-borne bacteria and the presence of Wolbachia phylotypes distinguished bacterial communities in female fleas from those in male fleas and in rodent blood. These results suggest that the overall abundance of a certain vector-borne microbe is more likely to be determined by the abundance of endosymbiotic bacteria in the vector, abundance of other vector-borne microbes co-occurring in the vector and in the host blood and by seasonal changes, than by host characteristics.     

The longevity of Leptopsylla segnis fleas (Siphonaptera: Leptopsyllidae)

In experiments, the mean life duration of fleas Leptopsylla segnis on white mice (abundance of fleas within natural limits, up to 10 fleas per mouse) was 22.7 days in females and 18.8 day in males. Maximum life duration was 51 and 37 days respectively. In cases, when the initial numbers of fleas were 20 and 28-34 fleas, the duration of life was decreased. The maximum limit decreased greater than the mean duration of life. A survival dynamics of fleas depended upon the flea number. It was found out, that in cases of high abundance of fleas in the beginning of experiments, the mortality rate of males was lower than in females. During the stay on a host the fleas lost gradually an ability to endure a starvation. Possible mechanisms of the regulation of flea abundance are discussed.     

Alternative prognostication of the population of fleas of the genus Xenopsylla (Siphonaptera)

A possibility of alternative prognostication of the autumn abundance of fleas was shown by means of statistical analysis of prognostication tables made up on the basis of the distribution of informative factors: indices of the abundance of the great gerbil, which is the main host of fleas, weather conditions and characteristics of the populations of the fleas.     

Characteristics of the multiplication of a virulent strain of the plague microbe in Xenopsylla cheopis fleas infected parenterally

Xenopsylla cheopis fleas infected parenterally with the virulent strain of plague microbe of gerbil variant preserved the agent to the end of their lives. In the body cavity the microbes retained their ability for reproduction which was, however, limited. During the first seven days after the infection the number of microbes slightly increased and later became stabilized. Its mean indices (mean g) varied within the limits of 500 to 2000 microbe cells per 1 individual, maximum index rarely exceeded 30 000 microbe cells. Parenteral infection with plague agent did not affect essentially the longevity of fleas.     

The Effect of Initial Infection of <Emphasis Type="Italic">Citellophilus tesquorum altaicus</Emphasis> Ioff, 1936 (Siphonaptera: Ceratophyllidae) with <Emphasis Type="Italic">Yersinia pestis</Emphasis> on Its Alimentary Activity,Mortality, and Biofilm Formation

Alimentary activity and mortality was assessed in fleas Citellophilus tesquorum altaicus non-infected with Yersinia pestis and those with initial infection levels 50 and 100% during feeding on a non-specific host (white mice). The presence of the plague pathogen in fleas significantly stimulated their feeding activity, especially in females. No effect of infection on flea mortality was observed. At the same time, male fleas died more frequently than females.     

不等单蚤和缓慢细蚤吸血前后脂肪和非特异性酯酶的组织化学研究

采用组织化学和显微摄影及定量图像分析技术,研究了不等单蚤Monopsyllus anisus (Rothschild)和缓慢细蚤Leptopsylla segnis (Schönherr) 在新羽化和吸血后发育过程中脂肪和非特异性酯酶的分布及活性。结果显示,两种新羽化蚤脂肪主要存在脂肪体中,非特异性酯酶则主要存在于唾液腺和神经链中。不等单蚤吸血后中肠脂滴增加,非特异性酯酶活性增强,随消化时间的延长脂滴逐渐减少,但不同消化时间非特异性酯酶活性差异不显著;而缓慢细蚤吸血后中肠未见或偶见少量脂滴,非特异性酯酶活性也有增强,但酶活性低于相同消化时间的不等单蚤。卵母细胞发育过程中脂滴增多,非特异性酯酶活性逐渐增强,且不等单蚤酶活性强于缓慢细蚤的。     

The influence of the host on the frequency of block formation in males and females of Citellophilus tesquorum altaicus Ioff, 1936 (Siphonaptera: Ceratophyllidae) and transmission of the plague pathogen by these fleas

Data obtained during feeding of infested females and males of Citellophilus tesquorum altaicus Ioff, 1936 (Siphonaptera: Ceratophyllidae), the main vector of plague in Tuva natural plague focus, on the natural hosts and on laboratory animals were analyzed. Sexual differences in fleas were found to depend on the host. Females fed more actively on longtailed ground squirrels Spermophilus undulatus than on white mice. The feeding activity of males on the two hosts did not differ. A higher mortality of infested females and males was observed during feeding on mice. The frequency of block formation and pathogen transmission in males was higher during bloodsucking on the ground squirrel; in females, during feeding on mice. Thus, sex-related differences in the plague pathogen transmission revealed in laboratory white mice may be not the same in nature. This should be taken into account when extrapolating the experimental data on the natural relations between the plague pathogen and ectoparasites.     

Paradoxopsyllus scorodumovi (Aphaniptera) flea, an effective vector of plague in a Gorno-Altai natural focus

A study was conducted on the fleas of P. scorodumovi and five local strains of the plague microbe, one of which is typical of the strains of the Altai subspecies and four are non-typical of this nidus. The fleas of this species are capable to transmit not only the plague agent of the strains typical of this nidus but also non-typical ones which differ in some biological properties and are avirulent for most carriers but Pallas's pika. Biological peculiarities of fleas of P. scorodumovi in addition to their high efficiency as vectors of the plague microbe enable us to associate the more active autumn epizooty with fleas of this species.     

First blood meal of Ctenocephalides canis (Siphonaptera: Pulicidae) on dogs: time to initiation of feeding and duration

Two experiments were conducted on dogs to evaluate interval to initiation and duration of the first blood meal of Ctenocephalides canis (Curtis). Percentage of fed male and female fleas was calculated for fleas held on dogs for 5, 15, 30, 60 min, 6, and 24 hr. Duration of first blood meal was also measured for individual fleas confined on dogs. When fleas were free in the hair coat, 21.2% had begun blood feeding within 5 min. After 1 hr, 72.5% of fleas had fed. After 6 hr, 95.2% of males and 100% of females had taken a blood meal, and 24 hr after deposition all fleas had fed. There was no significant difference between the 2 sexes. The mean delay between deposition and biting for fleas that began feeding within 15 min was 2 min 52 sec +/- 3 min 2 sec for female fleas and 3 min 8 sec +/- 2 min 45 sec for males. The mean duration of female and male meals was 5 min 3 sec +/- 3 min 41 sec and 6 min 9 sec +/- 6 min 8 sec, respectively. There was no significant difference between the 2 sexes. The dog flea took its blood meal on dogs more slowly than the cat flea did on cats; this meal was significantly longer for Ctenocephalides felis felis (Bouche) than for C. canis.     

Estimating the mean abundance and feeding rate of a temporal ectoparasite in the wild: Afrocimex constrictus (Heteroptera: Cimicidae)

The feeding frequency of blood-feeding invertebrates in the wild is largely unknown but is an important predictor for the potential of disease transmission and for estimating the effects blood feeding may have on the host population. We present a method to estimate the mean feeding frequency per individual parasite from the frequency distribution of fed and unfed individuals in the wild. We used three populations of the cimicid species, Afrocimex constrictus, that parasitises the fruit bat Rousettus aegyptiacus. We found that the area occupied by a bug refugium was a good predictor of the number of bugs in that refugia. The estimated parasite population sizes ranged from ca. 25,000 to 3 million bugs. Their mean abundance was 1-15 bugs per host individual. Preventing feeding by bugs in their natural habitat showed that bugs took approximately 20 days to return to an unfed stage. A formula is presented by which the distribution of digestion stages in the samples was used to calculate that A. constrictus feeds approximately every 7-10 days. The dry weight of a full blood meal was approximated as 13.3 mg. Therefore A. constrictus is estimated to draw an average of 1-28 microL blood per host per day. We suggest that any of our methods can be adjusted to be used in other haematophagous insects to estimate host and parasite population size, mean parasite abundance and blood meal size as well as mean feeding frequency in the wild, including the bed bug species that parasitise humans.     

Blood feeding and insulin-like peptide 3 stimulate proliferation of hemocytes in the mosquito Aedes aegypti

All vector mosquito species must feed on the blood of a vertebrate host to produce eggs. Multiple cycles of blood feeding also promote frequent contacts with hosts, which enhance the risk of exposure to infectious agents and disease transmission. Blood feeding triggers the release of insulin-like peptides (ILPs) from the brain of the mosquito Aedes aegypti, which regulate blood meal digestion and egg formation. In turn, hemocytes serve as the most important constitutive defense in mosquitoes against pathogens that enter the hemocoel. Prior studies indicated that blood feeding stimulates hemocytes to increase in abundance, but how this increase in abundance is regulated is unknown. Here, we determined that phagocytic granulocytes and oenocytoids express the A. aegypti insulin receptor (AaMIR). We then showed that: 1) decapitation of mosquitoes after blood feeding inhibited hemocyte proliferation, 2) a single dose of insulin-like peptide 3 (ILP3) sufficient to stimulate egg production rescued proliferation, and 3) knockdown of the AaMIR inhibited ILP3 rescue activity. Infection studies indicated that increased hemocyte abundance enhanced clearance of the bacterium Escherichia coli at lower levels of infection. Surprisingly, however, non-blood fed females better survived intermediate and high levels of E. coli infection than blood fed females. Taken together, our results reveal a previously unrecognized role for the insulin signaling pathway in regulating hemocyte proliferation. Our results also indicate that blood feeding enhances resistance to E. coli at lower levels of infection but reduces tolerance at higher levels of infection.     

Characterization of the salivary apyrase activity of three rodent flea species

1. Salivary gland lysates of the adult female fleas Oropsylla bacchi, Orchopea howardi and Xenopsylla cheopis hydrolyse ATP and ADP, but not AMP, thus characterizing the existence of a salivary apyrase activity. 2. In all species Mg++ or Ca++ function as activators, and a pH optimum between 7 and 8 is observed. 3. Salivary gland lysates of male fleas contain significantly smaller amounts of the enzyme activity than do those of female fleas. 4. Immediately following a blood meal, apyrase activity and protein content of female X. cheopis salivary glands are 2-3-fold less than that of unfed fleas, indicating that salivary apyrase activity is secreted during feeding. 5. It is suggested that, as in other arthropods, salivary apyrase may facilitate blood location and blood feeding by preventing ADP-induced platelet aggregation at the site of the bite.     

Effect of feeding frequency on the daily rhythms of acidic digestion in a teleost fish (gilthead seabream)

Gilthead seabream is a fish species of great importance in Mediterranean aquaculture, attracting many studies on nutrition and chronobiology, although nothing is known about the effect of feeding frequency on the daily rhythms of the gastric digestion process. In this article, we investigated daily rhythms in stomach fullness, gastric and intestine pH, as well as pepsin activity and expression of pepsinogen and proton pump in juvenile fish under three different feeding protocols: (A) one daily meal at 9:00, (B) two daily meals at 9:00 and 17:00 and (C) continuous feeding during the daytime. The results revealed that feeding protocol affected significantly the rhythm of gastric pH and the pepsin activity pattern. The gastric pH exhibited significant daily rhythms in the three cases with the acrophase located at night in the regimes A and B and during daytime, in the regime C. In the regimes A and B, the pepsin activity peaked few hours after the meals, although the afternoon meal in B produced a higher peak. In the regime C, the peak occurred in the middle of the feeding period. Lowest total pepsin activity was observed in regime A, and the highest activity with the regime C. In contrast, the pepsinogen gene expression remained low along the daily cycle, with an expression peak just before or after the morning meal in regimes A and C, respectively. The proton pump gene expression was also practically constant with a peak right after the morning meal in the regime C. On the other hand, intestinal pH showed a postprandial increase after the first morning meal in all the three treatments, recovering the resting values in the dark period. Two meals and continuous feeding allowed a better and prolonged gastric digestion and consequently the juveniles exhibited better growth with the same daily ration of food. In short, while the gastric digestion pattern is mainly driven by pH changes induced by the time of food ingestion, the regulation of the intestinal digestion seems to be more independent of the feeding protocol.     

Metabolic pathways for dietary lipids in the midgut of hematophagous Panstrongylus megistus (Hemiptera: Reduviidae)

The metabolism of dietary lipids in the anterior midgut of Panstrongylus megistus during blood digestion was studied. Fifth instar nymphs were fed a blood meal containing 7.1 +/- 0.4 mg of lipids, consisting mainly of triacylglycerol (TAG), and completed the overall process of digestion in about 20 days. Lipolysis of TAG and pathways for diacylglycerol (DAG) biosynthesis in the midgut were investigated by feeding the insects with [9,10-3H]-oleic acid-labeled triolein. Lumenal [3H]-triacylglycerol was hydrolyzed, generating mainly fatty acids (FA) and glycerol and to lesser extent, DAG. Almost no radioactivity associated with monoacylglycerol was found at any time. In midgut tissue, labeled fatty acids were incorporated into phosphatidic acid, DAG and TAG, whereas no significantly labeled monoacylglycerol was observed. In addition, the activities of enzymes related to DAG metabolism were assayed in non-blood fed midgut homogenates and at different times after feeding on a blood meal. Significant changes in the activities of phosphatidate phosphohydrolase (EC 3.1.3.4) and triacylglycerol lipase (EC 3.1.1.3) were observed during blood digestion, suggesting that these enzymes are important in regulating intracellular DAG synthesis and mobilization in midgut cells. Finally, the histological changes of lipid stores observed in anterior midgut confirmed the active process of uptake and trafficking of lipids performed by the enterocytes during blood digestion.     

The quantity of blood ingested by various species of fleas in an experiment

Data are given on the blood amount engorged by the fleas Xenopsylla conformis, X. cheopis, Leptopsylla segnis, Nosopsyllus laeviceps, N. consimilis, Ctenophthalmus golovi, Neopsylla setosa, Citellophilus tesquorum and Coptopsylla lamellifer. The average weight of blood portion in females of different species engorged for the first time ranged from 0.05 mg (X. conformis) to 0.72 mg (C. lamellifer). Females of most species, which had multiple blood meals, engorge more blood. Males engorge less blood than females and blood portions do not increase with age. In all sex-age groups most ectoparasites cease feeding spontaneously after having incomplete blood meal. Indices are suggested for the estimation of saturation rate in fleas during feeding.