The certainty that a species is accurately identified is the cornerstone of appearance based classification; however the methods used in classical taxonomy have yet to fully catch up with the digital age. Recognising this, the CO1 algorithm presented on the StripeSpotter platform was used to identify different species and sexes of mosquito wings (Diptera: Culicidae) and honey bee and bumblebee wings (Hymenoptera: Apidae). Images of different species of mosquito and bee wings were uploaded onto the CO1 database and test wing images were analysed to determine . CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/034819 doi: bioRxiv preprint first posted online Dec. 18, 2015; if this resulted in the correct species being identified. Out of a database containing 925 mosquito and bee wing images, the CO1 algorithm correctly identified species and sexes of test wing image presented, with a high degree of accuracy (80% to 100% depending on the species and database used, excluding sibling species) highlighting the usefulness of CO1 in identifying medically important as well as beneficial insect species. Using a larger database of wing images resulted in significantly higher numbers of test images being correctly identified than using a smaller database. The hind wings of Hymenoptera provided higher levels of correctly identified results than using the fore wings. The software should be used in conjunction with other identifying criteria (salient morphological features) in addition to the wings. CO1 is a powerful algorithm to use in identifying insect wings in its current form and has great potential if it is adapted and tailored for insect species identification. It is suggested that a primary aim in the digital age should be the production of a 'World Wide Database' of insect images, where all known insect images can be made available to everyone, with image recognition and species knowledge at its core.Introduction.
A number of image recognition systems have been specifically formulated for the individual recognition of large animals. These programs are versatile and can easily be adapted for the identification of smaller individuals such as insects. The Interactive Individual Identification System, I 3 S Classic, initially produced for the identification of individual whale sharks was employed to distinguish between different species of mosquitoes and bees, utilising the distinctive vein pattern present on insect wings. I 3 S Classic proved to be highly effective and accurate in identifying different species and sexes of mosquitoes and bees, with 80% to100% accuracy for the majority of the species tested. The sibling species Apis mellifera and Apis mellifera carnica were both identified with100% accuracy. Bombus terrestris terrestris and Bombus terrestris audax; were also identified and separated with high degrees of accuracy (90% to 100% respectively for the fore wings and 100% for the hind wings). When both Anopheles gambiae sensu stricto and Anopheles arabiensis were present in the database, they were identified with 94% and 100% accuracy respectively, allowing for a morphological and non-molecular method of sorting between these members of the sibling complex. Flat, not folded and entire, rather than broken, wing specimens were required for accurate identification. Only one wing image of each sex was required in the database to retrieve high levels of accurate results in the majority of species tested. The study describes how I 3 S was used to identify different insect species and draws comparisons with the use of the CO1 algorithm. As with CO1, I 3 S Classic proved to be suitable software which could reliably be used to aid the accurate identification of insect species. It is emphasised that image recognition for insect species should always be used in conjunction with other identifying characters in addition to the wings, as is the norm when identifying species using traditional taxonomic keys.
Novel insect identification techniques often lead to speculation on whether the method could cope with any intraspecific variation that might occur in a species.Using I 3 S Classic (Interactive Individual Identification System, Classic) and images of mosquito wings, different mosquito strains were tested with a copy of the strain present or absent from the database which contained images of other strains of the test species. When a wing image of the exact species, strain and sex was present in the database, there was 100% (or near 100%) retrieval of the correct species and strain at rank one. When the exact strain was absent from the database, but other strains of the same species were present, the retrieval rates at rank one were again high (100%) in the majority of cases and when they were not, the correct species was generally retrieved at rank two. Out of 40 different species and strains tested, only three were significantly different at rank one when the exact strain was absent from the database. In general, images of field strains selected for each other and therefore were similar to each other in greater numbers and instances than for the laboratory strains tested. When a copy of a strain was absent from the database, but other strains/sibling species were present, I 3 S retrieved the correct strains/sibling species at rank one in the majority of cases. In the one case of transgenic mosquitoes tested, I 3 S could reliably be used to identify transgenic mosquitoes from the parent stock as they were retrieved 100% at rank one when both the transgenic and unmodified parent strains were present in the database. This indicates the potential of using I 3 S to distinguish transgenic or other selectively bred strains from a parent strain, also selectively bred and wild mosquitoes, at least in the first phase after field release. Similarly, hybrid strains, sibling species and members of species complexes as in the Anopheles gambiae species complex could also be correctly identified when copies of all the relevant species/strains/siblings were in the database. This contradicts the belief that only molecular characterisation could separate A. gambiae s.s. from A. coluzzii, or A. arabiensis; I 3 S could accurately separate them all. I 3 S worked as it was set up to do, retrieving closely resembling images of the test insects from the database and ranking them in order of similarity.Dealing with any intraspecific variation was therefore not an issue if the software (I 3 S) was used systematically. I 3 S complements molecular and traditional taxonomic methods for species identification and the separation of sibling complexes and strains. In future, it should become the norm to maintain databases of insect wings and other body part images for use in image recognition.
Stress responses in insects can manifest as changes in size, shape and symmetry of the wings. Developing methods to measure and track such features could act as an early warning indicator of adverse events or, if all is well, provide assurance that field or laboratory colonies were fit, healthy and developing optimally. This is especially important in the case of newly developed transgenic insects, to assess morphology and as an indicator of their fitness. As body size and symmetry is known to be a significant correlate of fitness, the potential of transgenic insects is reflected in their phenotypic expression. Microsoft Paint and Photos as well as I3S Classic were used. The wings of transgenic mosquitoes DSM 1 & 2 were measured and compared to those of the parent population Anopheles gambiae G3. The right and left wings of both sexes were assessed to determine if they were symmetrical. Measurements indicated high wing symmetry in all the groups and sexes tested, indicating that the transgenic mosquitoes should be just as functional as their parents. The transgenic mosquitoes DSM 1 & 2 were found to be significantly larger in length and width than the parent population A. gambiae G3 and could be distinguished from the parent strain using I3S Classic software with 70 to 100% accuracy. I3S Classic ranked the correct sex of the test strain predominantly in the initial ranks indicating the differences in architecture of male and female wings. I3S Classic software was also used to assess wing symmetry. In keeping with the data from taking measurements, the software indicated that the wings were highly symmetrical, both the right and left wings of the correct strain were selected in the early first and second ranks in roughly equal measure. The importance of assessing the morphological characteristics of insects and of taking measurements during the investigative procedure was discussed.
Host age at infection has important implications for disease development. In mosquitoes, infections with microsporidia and later concurrent infections with malaria parasites, leads to a suppression in the development of malaria parasites. Host age at infection with microsporidia could have implications for disease outcomes when infection occurs subsequently with malaria parasites. Mosquito larvae can take between five to seven days or more depending on the temperature to reach the adult stage, giving the microsporidian Vavraia culicis, a theoretical head start in establishing and developing within larvae and possibly resulting in different levels of infection in emergent adult mosquitoes. To determine the effects of early or late infection with V. culicis, equal numbers of Anopheles coluzzii larvae were infected individually with a high or low dose of V. culicis, at different ages post hatching. Significantly fewer spores were produced from mosquitoes infected later, than ones infected earlier with microsporidia and there was an initial delay in the production of spores from later infected mosquitoes. In early infected larvae, there was no such initial delay and spore production took off unchecked. The infectious dose of V. culicis did not affect the total spore count per mosquito. Male mosquitoes produced fewer spores than females. Daily mosquito longevity and pupation was not affected significantly by infection, the infectious dose of V. culicis given or by the sex of the mosquito. Considering hourly deaths, early infected hosts died 17 to 18 hours earlier than later infected larvae. The number of V. culicis spores rose with increasing duration of infection. When equal duration of infection was considered, the findings remained the same. Host age at infection influences disease outcomes and virulence.
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