Changes in body size and fertility due to artificial and natural feeding of laboratory common bed bugs (Heteroptera: Cimicidae).

Rearing common bed bugs (Cimex lectularius L.) and other hematophagous insects is essential for basic, medical, and pest-control research. Logistically, acquiring fresh blood can be a challenge, while biologically, the eventual effects of different rearing and blood preparation protocols on bed bug genotype and phenotype pose a risk of biased research results. Using bed bug populations that are either bat- (BL) or human-related (HL), we tested the short- and long-term effects of rearing bugs on live bats or human volunteers, or artificially on CPDA (citrate phosphate dextrose, adenine)-treated blood, measuring meal size, body size, and fertility. We found that artificial feeding did not affect meal size compared with feeding on natural hosts. Long-term rearing across many generations of HL on CPDA-preserved blood led to reduced body size and fertility compared with populations reared on human volunteers. Blood preservatives increased the proportion of sterile eggs even after a single feed. Finally, our results indicated that laboratory reared bed bugs were smaller, regardless of the blood source, than wild bugs. Similar effects of artificial feeding or laboratory rearing alone should be considered in future studies using bed bug cultures to choose an appropriate rearing protocol. With regard to switching between bat and human hosts, HL took smaller meals and BL had lower fertility when fed on bats than when fed on humans. We attribute these results to methodological constrains, specifically the inconsistency of bat feeding, rather than to host specialization. Nevertheless, BL can be easily reared using human blood and artificial feeding systems.

Despite genetic isolation in sympatry, post-copulatory reproductive barriers have not evolved between bat- and human-associated common bedbugs (Cimex lectularius L.).

BACKGROUND: The common bedbug Cimex lectularius is a widespread ectoparasite on humans and bats. Two genetically isolated lineages, parasitizing either human (HL) or bat (BL) hosts, have been suggested to differentiate because of their distinct ecology. The distribution range of BL is within that of HL and bedbugs live mostly on synanthropic bat hosts. This sympatric co-occurrence predicts strong reproductive isolation at the post-copulatory level. RESULTS: We tested the post-copulatory barrier in three BL and three HL populations in reciprocal crosses, using a common-garden blood diet that was novel to both lineages. We excluded pre-copulation isolation mechanisms and studied egg-laying rates after a single mating until the depletion of sperm, and the fitness of the resulting offspring. We found a higher sperm storage capability in BL, likely reflecting the different seasonal availability of HL and BL hosts. We also observed a notable variation in sperm function at the population level within lineages and significant differences in fecundity and offspring fitness between lineages. However, no difference in egg numbers or offspring fitness was observed between within- and between-lineage crosses. CONCLUSIONS: Differences in sperm storage or egg-laying rates between HL and BL that we found did not affect reproductive isolation. Neither did the population-specific variation in sperm function. Overall, our results show no post-copulatory reproductive isolation between the lineages. How genetic differentiation in sympatry is maintained in the absence of a post-copulatory barrier between BL and HL remains to be investigated.

Effective NPM1 plasmid standards selection for minimal/measurable residual disease monitoring in acute myeloid leukemia.

BACKGROUND: NPM1 plasmid standards are required for absolute quantification of minimal residual disease in acute myeloid leukemia patients. The standards are usually obtained, next to commercially constructed gene fragments, from transgenic bacteria colonies. However, this procedure is laborious and very time consuming. METHODS AND RESULTS: We have developed a PCR method that speeds up, simplifies, and streamlines the process of preparing NPM1 plasmid standards. The method is based on a combination of three primers, two surrounding the usual NPM1 mutation position and one over the mutation site. With this method, we were able to clearly distinguish plasmids with at least 15 different NPM1 mutations from the wild-type NPM1 plasmid. CONCLUSIONS: With the new approach, preparing NPM1 plasmid standards is easier, identifying NPM1-positive colonies is possible in less than a day and moreover, for a lower price than commercially constructed gene fragments.

Seminal fluid and sperm diluent affect sperm metabolism in an insect: Evidence from NAD(P)H and flavin adenine dinucleotide autofluorescence lifetime imaging.

Sperm metabolism is fundamental to sperm motility and male fertility. Its measurement is still in its infancy, and recommendations do not exist as to whether or how to standardize laboratory procedures. Here, using the sperm of an insect, the common bedbug, Cimex lectularius, we demonstrate that standardization of sperm metabolism is required with respect to the artificial sperm storage medium and a natural medium, the seminal fluid. We used fluorescence lifetime imaging microscopy (FLIM) in combination with time-correlated single-photon counting (TCSPC) to quantify sperm metabolism based on the fluorescent properties of autofluorescent coenzymes, NAD(P)H and flavin adenine dinucleotide. Autofluorescence lifetimes (decay times) differ for the free and protein-bound state of the co-enzymes, and their relative contributions to the lifetime signal serve to characterize the metabolic state of cells. We found that artificial storage medium and seminal fluid separately, and additively, affected sperm metabolism. In a medium containing sugars and amino acids (Grace's Insect medium), sperm showed increased glycolysis compared with a commonly used storage medium, phosphate-buffered saline (PBS). Adding seminal fluid to the sperm additionally increased oxidative phosphorylation, likely reflecting increased energy production of sperm during activation. Our study provides a protocol to measure sperm metabolism independently from motility, stresses that protocol standardizations for sperm measurements should be implemented and, for the first time, demonstrates that seminal fluid alters sperm metabolism. Equivalent protocol standardizations should be imposed on metabolic investigations of human sperm samples.

Male diet affects female fitness and sperm competition in human- and bat-associated lineages of the common bedbug, Cimex lectularius.

Sperm performance can vary in ecologically divergent populations, but it is often not clear whether the environment per se or genomic differences arising from divergent selection cause the difference. One powerful and easily manipulated environmental effect is diet. Populations of bedbugs (Cimex lectularius) naturally feed either on bat or human blood. These are diverging genetically into a bat-associated and a human-associated lineage. To measure how male diet affects sperm performance, we kept males of two HL and BL populations each on either their own or the foreign diet. Then we investigated male reproductive success in a single mating and sperm competition context. We found that male diet affected female fecundity and changed the outcome of sperm competition, at least in the human lineage. However, this influence of diet on sperm performance was moulded by an interaction. Bat blood generally had a beneficial effect on sperm competitiveness and seemed to be a better food source in both lineages. Few studies have examined the effects of male diet on sperm performance generally, and sperm competition specifically. Our results reinforce the importance to consider the environment in which sperm are produced. In the absence of gene flow, such differences may increase reproductive isolation. In the presence of gene flow, however, the generally better sperm performance after consuming bat blood suggests that the diet is likely to homogenise rather than isolate populations.

NSC348884 cytotoxicity is not mediated by inhibition of nucleophosmin oligomerization.

Nucleophosmin (NPM) mutations causing its export from the nucleoli to the cytoplasm are frequent in acute myeloid leukemia (AML). Due to heterooligomerization of wild type NPM with the AML-related mutant, the wild-type becomes misplaced from the nucleoli and its functions are significantly altered. Dissociation of NPM heterooligomers may thus restore the proper localization and function of wild-type NPM. NSC348884 is supposed to act as a potent inhibitor of NPM oligomerization. The effect of NSC348884 on the NPM oligomerization was thoroughly examined by fluorescence lifetime imaging with utilization of FRET and by a set of immunoprecipitation and electrophoretic methods. Leukemia-derived cell lines and primary AML cells as well as cells transfected with fluorescently labeled NPM forms were investigated. Our results clearly demonstrate that NSC348884 does not inhibit formation of NPM oligomers neither in vivo nor in vitro. Instead, we document that NSC348884 cytotoxicity is rather associated with modified cell adhesion signaling. The cytotoxic mechanism of NSC348884 has therefore to be reconsidered.

Nucleophosmin in leukemia: Consequences of anchor loss.

Nucleophosmin (NPM), one of the most abundant nucleolar proteins, has crucial functions in ribosome biogenesis, cell cycle control, and DNA-damage repair. In human cells, NPM occurs mainly in oligomers. It functions as a chaperone, undergoes numerous interactions and forms part of many protein complexes. Although NPM role in carcinogenesis is not fully elucidated, a variety of tumor suppressor as well as oncogenic activities were described. NPM is overexpressed, fused with other proteins, or mutated in various tumor types. In the acute myeloid leukemia (AML), characteristic mutations in NPM1 gene, leading to modification of NPM C-terminus, are the most frequent genetic aberration. Although multiple mutation types of NPM are found in AML, they are all characterized by aberrant cytoplasmic localization of the mutated protein. In this review, current knowledge of the structure and function of NPM is presented in relation to its interaction network, in particular to the interaction with other nucleolar proteins and with proteins active in apoptosis. Possible molecular mechanisms of NPM mutation-driven leukemogenesis and NPM therapeutic targeting are discussed. Finally, recent findings concerning the immunogenicity of the mutated NPM and specific immunological features of AML patients with NPM mutation are summarized.

AML-associated mutation of nucleophosmin compromises its interaction with nucleolin.

C-terminal mutations of the nucleolar protein nucleophosmin (NPM) are the most frequent genetic aberration detected in acute myeloid leukemia (AML) with normal karyotype. The mutations cause aberrant cytoplasmic localization of NPM and lead to loss of functions associated with NPM nucleolar localization, e.g. in ribosome biogenesis or DNA-damage repair. NPM has many interaction partners and some of them were proved to interact also with the mutated form (NPMmut) and due to this interaction thereby to be withdrawn from their site of action. We analyzed the impact of the mutation on NPM interaction with nucleolin (NCL) which is also prevalently localized into the nucleolus and cooperates with wild-type NPM (NPMwt) in many cellular processes. We revealed that the NCL-NPM complex formation is completely abolished by the mutation and that the presence/absence of the interaction is not affected by drugs causing genotoxic stress or differentiation. Deregulation resulting from changes of NCL/NPMwt ratio may contribute to leukemogenesis.